Ethnopharmacological Uses, Phytochemistry, and Pharmacological Properties of Croton macrostachyus Hochst. Ex Delile: A Comprehensive Review

Evid Based Complement Alternat Med. 2017; 2017: 1694671. Published online 2017 Oct 12. doi: 10.1155/2017/1694671 PMCID: PMC5660826 Alfred Maroyi Medicinal Plants and Economic Development (MPED) Research Center, Department of Botany, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa. Article notes ► Copyright and License information ► Go to: Abstract Croton macrostachyus is widely used as herbal medicine by the indigenous people of tropical Africa. The potential of C. macrostachyus as herbal medicine, the phytochemistry, and pharmacological properties of its parts used as herbal medicines are reviewed. The extensive literature survey revealed that C. macrostachyus is traditionally used to treat or manage at least 81 human and animal diseases and ailments. The species is used as herbal medicine for diseases and ailments such as abdominal pains, cancer, gastrointestinal disorders, malaria, pneumonia, sexually transmitted infections, skin infections, typhoid, and wounds and as ethnoveterinary medicine. Multiple classes of phytochemicals such as alkaloids, amino acids, anthraquinones, carbohydrates, cardiac glycosides, coumarins, essential oil, fatty acids, flavonoids, phenolic compounds, phlobatannins, polyphenols, phytosteroides, saponins, sterols, tannins, terpenoids, unsaturated sterol, vitamin C, and withanoides have been isolated from the species. Pharmacological studies on C. macrostachyus indicate that it has a wide range of pharmacological activities such as anthelmintic, antibacterial, antimycobacterial, antidiarrhoeal, antifungal, anticonvulsant and sedative, antidiabetic, anti-inflammatory, antileishmanial, antioxidant, antiplasmodial, and larvicidal effects. Croton macrostachyus has potential as a possible source of a wide range of pharmaceutical products for the treatment of a wide range of both human and animal diseases and ailments. Go to: 1. Introduction Croton macrostachyus Hochst. ex Delile is a species of the genus Croton L., Euphorbiaceae family, commonly known as the spurge family. Croton macrostachyus is a medium sized, drought-deciduous pioneer tree which regenerates naturally in less productive sites including forest edges, mountain slopes, and waste grounds under a wide range of ecological conditions [1–3]. Croton macrostachyus is regarded as a multipurpose tree by subsistence farmers in Ethiopia, Kenya, and Tanzania [3–6], as it is often grown and managed in home gardens for provision of several ecosystem goods and services. In Ethiopia, for example, C. macrostachyus is a major tree intercropped in agroecosystems in order to increase soil productivity in midaltitude and semiarid areas [7]. There is also tremendous interest in the medicinal uses and pharmacological properties of C. macrostachyus throughout its distributional range in tropical Africa [8–11]. Research by these authors revealed that C. macrostachyus is an important medicinal plant in tropical Africa with potential of providing important pharmaceutical products to be used by rural and urban communities who rely on herbal medicines for primary healthcare. Integration of traditional medicine and modern medicine has been recommended by the World Health Organization (WHO) since 1978 [12], mainly because traditional medicines are perceived to be more affordable, accessible, and acceptable to poor rural and urban communities and those living in marginalized areas [13]. Considering the documented ethnomedicinal uses of C. macrostachyus in tropical Africa [8–11], certainly the species has potential in playing an important role in the primary healthcare of communities throughout its distributional range. It is therefore important to assess if there is correlation between the ethnomedicinal uses of C. macrostachyus and the recent documented phytochemical and pharmacological properties of the species. Therefore, the present review collates the fragmented information on traditional uses, phytochemistry, pharmacology, and toxicology of the species. It is hoped that this information will highlight the importance of C. macrostachyus as a potential source of a wide range of pharmaceutical products in tropical Africa and will provide a new direction for researchers in the future. Go to: 2. Methodology of the Review Croton macrostachyus and other historical names and synonyms of the species were used as the keywords in searching the major databases including Web of Science, Scopus, Google Scholar, Science Direct, BioMed Central (BMC), PubMed, and Springerlink documenting traditional uses, medicinal uses, ethnobotany, ethnomedicinal uses, ethnopharmacology, pharmacology, phytochemistry, and therapeutic value of the species. Additional literature, including preelectronic literature such as dissertations, theses, and other grey materials were sourced from the University of Fort Hare library in South Africa. Go to: 3. Botanical Profile, Taxonomy, and Distribution of Croton macrostachyus The genus name “Croton” was derived from a Greek word “kroton,” a tick, referring to thick smooth seeds, a common feature of most Croton species which belong to the Crotonoideae subfamily of the Euphorbiaceae family [14]. The specific name “macrostachyus” is a contraction of two words, the Greek word “macro” meaning large and “stachyus” relating to the spike, hence, a species characterized by large spikes [15]. Historical names or synonyms of C. macrostachyus are C. acuminatus R. Br., C. butaguensis De Wild., C. guerzesiensis Beille ex A. Chev., C. macrostachyus var. mollissimus Chiov., Oxydectes macrostachya (Hochst. ex Delile) Kuntze, and Rottlera schimperi Hochst. & Steud. (http://www.theplantlist.org/tpl1.1/record/kew-50457). Croton macrostachyus is commonly known as “broad-leaved croton” or “rush foil” in English, “bisana” in Amharic in Ethiopia, and “msinduzi” in Swahili in east Africa [11, 16–24]. Croton macrostachyus is widely distributed in tropical Africa, from Guinea east to Ethiopia and Somalia, south to Angola, Mozambique, and Madagascar (Figure 1). The species has been reported to occur in Angola, Burundi, Cameroon, Central African Republic, Democratic Republic of Congo (DRC), Ethiopia, Ghana, Guinea, Ivory Coast, Kenya, Madagascar, Malawi, Mozambique, Nigeria, Rwanda, Somalia, South Sudan, Sudan, Tanzania, Uganda, and Zambia [3, 25]. According to Mairura [11], C. macrostachyus is common in secondary forests, especially on forest edges and along rivers or lakes, in moist or dry evergreen upland forest, woodland, wooded grassland, bushland, and along roadsides, often on soils of volcanic origin at altitude between 200 to 3400 m above sea level and mean annual rainfall between 150 mm and 1200 mm. Figure 1 Figure 1 Distribution of Croton macrostachyus in tropical Africa. Croton macrostachyus is a monoecious or dioecious, deciduous, medium sized tree up to 30 m tall [2]. The bole is cylindrical up to 100 cm in diameter with grey to grey-brown bark, finely fissured, and cracked, inner bark pale brown to reddish brown with a peppery smell [1]. The leaves are alternate, simple, turning orange before falling with linear stipules, up to 15 mm long [1]. The petiole is up to 20 cm long with two stalked glands at top [2]. The leaf blade is ovate-elliptical to almost circular, up to 25 cm × 20 cm in size with a cordate base and acuminate apex acuminate with irregularly toothed margins, densely stellate hairy on both sides, and whitish green beneath [1]. The inflorescence is a slender, terminal raceme up to 35 cm long, either with only male or female flowers or male and female flowers variably mixed [2]. Male flower has a pedicel which is 3–10 mm long with campanulate calyx with ovate to triangular lobes, 2.5–3.5 mm long with densely white hairy margins [1]. The petals are oblong to oblanceolate, 3–4.5 mm long and have 15–17 free stamens [1]. The female flower has a pedicel which is 2–4 mm long, fleshy, calyx as in male flowers but lobes more triangular, persistent in fruit, petals linear, or absent up to 1.5 mm long [1]. The ovary is superior, rounded, densely stellate hairy with three styles, 3–6 mm long, twisted, and curved [2]. The fruit is a globular capsule, 8–12 mm in diameter with a centrally depressed apex, whitish to pale greyish brown in colour. The seeds are ellipsoid, 6–8 mm × 4–5.5 mm in size, flattened, and cream-coloured [1]. Go to: 4. Traditional and Contemporary Uses of Croton macrostachyus The bark, fruits, leaves, roots, and seeds of C. macrostachyus are reported to possess diverse medicinal properties and cure various human and animal diseases and ailments throughout the distributional range of the species (Table 1). Croton macrostachyus is used as herbal medicine for at least 61 and 20 human and animal diseases and ailments, respectively (Table 1). There is cross-cultural agreement among ethnomedicinal uses of C. macrostachyus throughout its distributional range, and there is also a high degree of medicinal use consensus (recorded in at least two countries) for bleeding, blood clotting, cancer, constipation, diarrhoea, epilepsy, malaria, pneumonia, purgative, ringworm, skin diseases or infections, stomach ache, typhoid, worm expulsion, and wounds (see Table 1). Table 1 Table 1 Ethnomedicinal uses of Croton macrostachyus in tropical Africa. In Cameroon, Ethiopia, Kenya, Rwanda, Somalia, Tanzania, and Uganda, leaf decoction, infusion or maceration, stem bark, or root bark of C. macrostachyus is taken as a purgative and vermifuge (Table 1). Croton macrostachyus is also used in combination with other plant species. For example, in Ethiopia, a bark decoction of C. macrostachyus is often mixed with roots of Cucumis ficifolius A. Rich. as remedy for abdominal pain [28]. Leaf decoction of C. macrostachyus is often mixed with leaves of Trichilia spp. and Rhamnus prinoides L'Hérit. as remedy for diarrhoea and dysentery in humans and to repel external parasites in livestock [48]. Research by Mesfin et al. [60] and Bekele and Reddy [61] revealed that crushed leaves of C. macrostachyus are boiled in water mixed with Allium sativum L. bulb roasted with butter; the concoction is allowed to brew overnight and taken orally the following day as remedy for malaria. For skin diseases and wounds, the fruit decoction of C. macrostachyus is mixed with leaves of Hagenia abyssinica (Bruce) J.F.Gmel. and applied topically on affected body parts [69] while leaf sap of C. macrostachyus is mixed with coconut (Cocos nucifera L.) milk and applied on wounds [44] or a leaf decoction mixture of C. macrostachyus, Cynoglossum lanceolatum Forssk., and Dodonaea viscosa subsp. angustifolia (L.f.) J.G.West is applied on wounds [17]. According to Teklehaymanot et al. [17], bark decoction of C. macrostachyus is mixed with stems of Glinus lotoides L. as herbal medicine for tapeworms while bark decoction of C. macrostachyus is mixed with leaves of Juniperus procera Hochst. ex Endl., stems of Eragrostis tef (Zucc.) Trotter, and roots of Cyphostemma cyphopetalum (Fresen.) Desc. ex Wild & Drummond, Solanum anguivi Lam., and Solanum marginatum L. f. as herbal medicine for rabies in Ethiopia. Research by Nahayo et al. [72] revealed that leaf decoction of C. macrostachyus mixed with leaves of Baccharoides calvoana subsp. meridonalis (Wild) Isawumi, El-Ghazaly & B.Nord, Vernonia auriculifera Hiern, and Salvia nilotica Juss. ex Jacq. or bark decoction of C. macrostachyus mixed with bark of Morella salicifolia subsp. mildbraedii (Engl.) Verdc. & Polhill, Olea capensis subsp. macrocarpa (C.H.Wright) Verdc., and Elaeis guineensis Jacq. is used for worm expulsion in Rwanda. The leaves of C. macrostachyus are used by farmers in Kenya as biological pest control when mixed with tobacco (Nicotiana tobacuum L.) and boiled overnight [57]. The resultant mixture is used as a biological pesticide for the control of maize stalk borers and aphids [57]. Table 1 provides a summary of ethnomedicinal uses and plant parts of C. macrostachyus used among diverse ethnic groups in tropical Africa. In Cameroon, root decoction of C. macrostachyus is used as purgative [65], while bark and leaf decoctions are used as remedies for epilepsy, insomnia, and typhoid [50, 63, 74]. In Ethiopia, C. macrostachyus has many uses including abdominal pain, abortifacient, amoebiasis, antidote for scorpion, and snake venom, anthrax, ascariasis, cancer, constipation, diarrhoea, dysentery, epilepsy, jaundice, leprosy, malaria, ringworm, sexually transmitted infections (STIs), skin diseases, stomach ache, tapeworms, typhoid, and wounds [17, 20, 26, 28–32, 34, 35, 38, 40, 44, 45, 49–52, 55–57, 60, 61, 63, 65, 67, 69, 72, 74, 82]. In Kenya, C. macrostachyus bark juice, leaf, and root decoction is used as remedy for backache, bleeding, cancer, colds, cough, diarrhoea, dysmenorrhoea, east coast fever, malaria, measles, obesity, pneumonia, ringworm, skin diseases, typhoid, warts, and wounds [37, 39, 46, 58, 59, 62, 68]. Research by Mazzanti et al. [66] revealed that seeds are used as purgatives in Somalia. In Tanzania, fruit and decoction of C. macrostachyus are used as purgative [16], while fruit, leaf, and root decoctions are used as remedies for abdominal pain, constipation, diabetes, ringworm, skin infections, sores, and worm expulsion [16, 27, 43]. In Uganda, C. macrostachyus bark and root decoctions are used as remedies for headache, stomach ache, and worms [54, 71]. Bark, leaf, root, and twig decoctions of C. macrostachyus are used as ethnoveterinary medicine in Ethiopia and Kenya for abdominal pain, blackleg, bleeding, bloat, colic, constipation, dermatophilosis, epilepsy, fever, rabies, rectum prolapsed, ringworm, scabies, skin diseases, warts, and wounds [17, 21, 23, 35, 36, 38, 44, 48, 68, 75–78]. In Kenya, Tanzania, and Uganda, C. macrostachyus is commonly planted as an ornamental or shade tree in villages and the tree is also used as a shade-bearer on coffee plantations and other crops [16, 57, 80]. The wood is used in Cameroon, Ethiopia, Kenya, Tanzania, and Uganda to make tool handles, small stools, boxes, crates, and plywood, as flooring and building material and in carpentry [16, 40, 57, 63, 65, 80]. The wood is used as fuel that burns even when green but produces a rather unpleasant spicy odour and much smoke; it is also used to make charcoal [11, 16, 63, 65, 80]. Due to its drought hardiness and fast growth, Croton macrostachyus is considered useful for afforestation of shifting sand dunes, degraded waste land, hill slopes, ravines, and lateritic soils [11]. Go to: 5. Phytochemistry Multiple classes of phytochemicals including alkaloids, amino acids, anthraquinones, carbohydrates, cardiac glycosides, coumarins, essential oil, fatty acids, flavonoids, phenolic compounds, phlobatannins, polyphenols, phytosteroides, saponins, sterols, tannins, terpenoids, unsaturated sterol, vitamin C, and withanoides have been identified from C. macrostachyus fruits, leaves, stem bark, and twigs [24, 30, 47, 55, 83–89]. Addae-Mensah et al. [83] isolated betulin 1, lupeol 2, crotepoxide 3, β-sitosterol 4, and stigmasterol 5 from stem barks and twigs of C. macrostachyus (see Table 2, Figure 2). Kapingu et al. [90] isolated 3β-acetoxy taraxer-14-en-28-oic acid 6, trachyloban-19-oic acid 7, trachyloban-18-oic acid 8, neoclerodan-5,10-en-19,6β; 20,12-diolide 9, 3α,19-dihydroxytrachylobane 10, and 3α,18,19-trihydroxytrachylobane 11 from roots of C. macrostachyus. Tane et al. [91] isolated crotepoxide 3 and crotomacrine 12 from fruits of C. macrostachyus. Tene et al. [92] isolated betulin 1, lupeol 2, floridolide A 13, hardwickic acid 14, and 12-oxo-hardwickic acid 15 from stem bark of C. macrostachyus. Tala et al. [93] isolated and identified betulin 1, lupeol 2, β-sitosterol 4, stigmasterol 5, lupenone 16, betulinic acid 17, 28-O-acetylbetulin 18, lupeol acetate 19, zeorin 20, benzoic acid 21, methyl gallate 22, methyl 2,4-dihydroxy-3,6-dimethylbenzoate 23, lichexanthone 24, and β-sitosterol palmitate 25 from twigs of C. macrostachyus. Figure 2 Figure 2 Chemical structures of major compounds isolated from leaves, roots, and stems of Croton macrostachyus. Table 2 Table 2 Chemical compounds isolated and characterized from Croton macrostachyus. Go to: 6. Pharmacological Activities A number of pharmacological activities of C. macrostachyus have been reported in literature justifying some of its ethnomedicinal uses listed in Table 1. These pharmacological activities include anthelmintic [30, 95], antibacterial [53, 55, 62, 85, 92, 96–100], anticonvulsant and sedative [50], antidiabetic [88], antidiarrhoeal [24], antifungal [62, 86, 92, 101], anti-inflammatory [84, 102], antileishmanial [94], antioxidant [86], antiplasmodial [87, 89, 103, 104], antimycobacterial [105], larvicidal [19, 50], and cytotoxicity [27, 43, 73, 96, 104–108]. 6.1. Anthelmintic Eguale et al. [30] evaluated anthelmintic activities of crude aqueous and hydroalcoholic extracts of the seeds of C. macrostachyus on eggs and adult tapeworms (Haemonchus contortus). Both aqueous and hydroalcoholic extracts of C. macrostachyus induced statistically significant egg hatching inhibition (p < 0.05) with aqueous extract requiring maximum concentration of 0.5 mg/ml to induce 100% egg hatch inhibition while the hydroalcoholic extracts did not induce complete inhibition at highest concentration tested of 2 mg/ml. The aqueous extract of C. macrostachyus induced 50% inhibition (ED50) at 0.10 mg/ml which was at a lower concentration than the hydroalcoholic extract at ED50 value of 0.32 mg/ml [30]. After 24 hours of exposure of adult Haemonchus contortus to different concentration of plant extracts, hydroalcoholic extracts of the species produced mortality of adult Haemonchus contortus to the level of 90% at concentration of 8 mg/ml while aqueous extract produced only 36.67% at the same concentration [30]. Similarly, Aleme et al. [95] evaluated the anthelmintic effects of crude aqueous extracts of the leaves of C. macrostachyus against adults of live Haemonchus contortus and the efficacy of these crude aqueous extracts was determined based on the mortality rate of the adult parasite. The efficacy at 4 mg/ml of the aqueous extracts of C. macrostachyus was 70% against the adult stage of Haemonchus contortus and the efficacy of the positive control, albendazole, against the adult parasite was dose-dependent and all the adult worms were dead at a concentration of 0.5 mg/ml within 24 hours [95]. These findings indicate that C. macrostachyus has potential anthelmintic effect and could be used as an inexpensive and eco-friendly alternative to controlling tapeworm infections. 6.2. Antibacterial Geyid et al. [55] evaluated antibacterial activities of methanol, petroleum ether, and aqueous fruit extracts of C. macrostachyus against Bacillus cereus, Escherichia coli, Neisseria gonorrhoea, Salmonella typhi, Salmonella typhimurium, Shigella dysentery, Shigella flexineri, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes using the agar dilution method. Methanol fruit extract of C. macrostachyus inhibited growth of Neisseria gonorrhoeae at four levels of concentration, that is, 250, 500, 1000, and 2000 μg/ml. Mesfin et al. [85] evaluated antibacterial activities of chloroform, n-butanol, and aqueous fractions of C. macrostachyus against Neisseria gonorrhoeae using the agar dilution method. Chloroform and n-butanol fractions of C. macrostachyus were more active with minimum inhibitory concentration (MIC) values ranging from 125 to 250 μg/mL [85]. Wagate et al. [96] also evaluated antibacterial activities of leaf and root methanolic extracts of C. macrostachyus against Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus using the broth dilution method with benzylpenicillin and streptomycin as positive controls. The methanolic extracts of C. macrostachyus were the most active against Bacillus cereus with minimum inhibitory concentration (MIC) value of 15.6 mg/mL and MIC value of 250 mg/mL against both Escherichia coli and Pseudomonas aeruginosa [96]. In another study, Wagate et al. [97] evaluated antibacterial activities of methanol leaf and root extracts of C. macrostachyus against Bacillus cereus, Escherichia coli, Micrococcus lutea, and Pseudomonas aeruginosa using broth dilution method with benzylpenicillin and streptomycin as positive controls. Croton macrostachyus methanol leaf and root extracts were active against Bacillus cereus with minimum inhibitory concentration (MIC) value of 15.6 mg/mL as well as Escherichia coli and Pseudomonas aeruginosa, both with MIC value of 250 mg/mL [97]. Tene et al. [92] also evaluated antibacterial activities of the ethanol stem bark extract of C. macrostachyus and five compounds isolated from the stem bark of the species, namely, betulin 1, lupeol 2, floridolide A 13, hardwickic acid 14, and 12-oxo-hardwickic acid 15 against Klebsiella pneumoniae, Salmonella typhi, and Staphylococcus aureus. The ethanol stem bark extract demonstrated some activity with the minimum bactericidal concentration (MBC) values ranging from 31.25 to 500 μg/ml; the best activity of 31.25 μg/ml was against Staphylococcus aureus. The compounds demonstrated some activity with the minimum bactericidal concentration (MBC) values ranging from 15.62 to >1000 μg/ml. The best activity was demonstrated by betulin 1 with MIC value of 15.25 μg/ml against Salmonella typhi and Staphylococcus aureus, and MIC value of 31.25 μg/ml against Klebsiella pneumoniae, while hardwickic acid 14 had MIC value of 31.25 μg/ml against Staphylococcus aureus and 12-oxo-hardwickic acid 15 had MIC value of 62.5 μg/ml against Staphylococcus aureus [92]. Belay et al. [98] evaluated antibacterial activities of volatile fractions of C. macrostachyus fruits against Bacillus cereus, Citrobacter spp., Escherichia coli, Klebsiella pneumonia, Listeria monocytogenes, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella paratyphi, Shigella dysenteriae, Staphylococcus aureus, and Streptococcus pyogenes using dimethyl sulfoxide (DMSO) and Tween-80 as negative controls and gentamicin as positive control. Antibacterial activity was demonstrated with minimum inhibitory concentration (MIC) values ranging from 0.1 to 12.5 μg/ml and minimum bactericidal concentration (MBC) values ranging from 0.2 to 25 μg/ml [98]. Taye et al. [99] also evaluated antibacterial activities of aqueous and methanol leaf extracts of C. macrostachyus against Escherichia coli, Proteus vulgaris, Pseudomonas aeuruginosa, Staphylococcus aureus, and Streptococcus pyogenes using the agar well diffusion method with ciprofloxacin and amoxicillin as positive controls. Antibacterial activity was demonstrated by methanol leaf extract against Streptococcus pyogenes with minimum bacterial concentration (MBC) value of 7.81 mg/mL. Mesfin et al. [53] also evaluated antibacterial activities of chloroform, n-butanol, and aqueous leaf extracts of C. macrostachyus against Neisseria gonorrhoeae using the agar dilution method. Chloroform and n-butanol fractions were identified to be more active with minimum inhibitory concentration (MIC) values between 125 and 250 μg/ml [53]. Similarly, Sendeku et al. [100] evaluated antibacterial activities of chloroform, ethanol and methanol leaf extracts of C. macrostachyus using the agar well diffusion and broth dilution assay methods against Escherichia coli, Klebsiella pneumonia, Salmonella pneumonia, Shigella flexneri, and Staphylococcus aureus. The leaf extract showed some activity with minimum inhibitory concentrations (MIC) varying from 3.75 to 30.0 mg/ml and minimum bactericidal concentrations (MBC) varying from 7.5 to 40.0 mg/ml [100]. Recently, Obey et al. [62] evaluated antibacterial activities of methanol, ethyl acetate, and butanol stem bark extracts and purified lupeol 2 isolated from C. macrostachyus against Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, and Listeria monocytogenes using the agar well diffusion method. The most promising broad scale antibacterial activity against all the studied pathogens was shown by the ethyl acetate extract with minimum inhibitory concentrations (MICs) ranging from 125 to 250 mg/mL and lupeol 2 had the lowest MIC value of 125 mg/mL against Klebsiella pneumoniae. These antibacterial activities displayed by different extracts [53, 55, 62, 85, 92, 96–100] somehow confirm the potential of C. macrostachyus in the treatment and management of bacterial infections as detailed in Table 1. 6.3. Antimycobacterial Gemechu et al. [105] evaluated antimycobacterial activities of methanolic leaf extracts of C. macrostachyus against Mycobacterium tuberculosis and Mycobacterium bovis strains using 96 wells of microplate with the help of visual Resazurin Microtiter assay. The methanolic leaf extracts of C. macrostachyus demonstrated antimycobacterial activity with minimum inhibitory concentration (MIC) values ranging from 12.5 to 100 μg/mL. The results of this study demonstrate that C. macrostachyus has potential as herbal medicine in the treatment and management of tuberculosis, a leading cause of death in sub-Saharan Africa [109]. However, further investigations are needed aimed at identifying chemical constituents of C. macrostachyus responsible for these activities and their mode of action. 6.4. Antifungal Tene et al. [92] evaluated antifungal activities of the ethanol stem bark extract of C. macrostachys and chemical compounds isolated from the stem bark of the species, namely, betulin 1, lupeol 2, floridolide A 13, hardwickic acid 14, and 12-oxo-hardwickic acid 15 against Candida albicans, Candida krusei, and Cryptococcus neoformans. The ethanol stem bark extract demonstrated some activity with the minimum fungicidal concentration (MFC) values ranging from 62.5 to 1000 μg/ml, and the best activity of 62.5 μg/ml was against Candida albicans. The compounds demonstrated some activity with the minimum fungicidal concentration (MFC) values ranging from 7.81 to >1000 μg/ml. The best activity was demonstrated by 12-oxo-hardwickic acid 15 with MIC value of 7.81 μg/ml against Candida albicans, MIC value of 31.25 μg/ml against Candida krusei and Cryptococcus neoformans, and hardwickic acid 14 had MIC value of 62.5 μg/ml against Candida albicans [92]. Abera et al. [101] also evaluated antifungal activity of aqueous and ethanol extracts of C. macrostachyus against Colletotrichum kahawae, a fungus that causes coffee berry disease. Croton macrostachyus aqueous and ethanol extracts reduced radial growth of Colletotrichum kahawae in ethanol and aqueous extracts by 68% and 88%, respectively. This study indicated the possible use of C. macrostachyus extracts as an alternative means of coffee berry disease management [101]. Teugwa et al. [86] evaluated antifungal activities of aqueous and methanol leaf extracts of C. macrostachyus against Trichophyton rubrum, Trichophyton soudanense, and Trichophyton violaceum using the agar dilution method with amphotericin as positive control. All extracts tested showed antifungal activity against the three Trichophyton species tested, with minimum inhibitory concentration (MIC) and minimum fungicidal concentrations (MFC) varying from 17.50 to 27.50 and 20 to 30 mg/ml, respectively. Recently, Obey et al. [62] evaluated antifungal activities of methanol, ethyl acetate, and butanol stem bark extracts and purified lupeol 2 isolated from C. macrostachyus against Candida albicans using the agar well diffusion method. The most promising broad scale antifungal activity was shown by the ethyl acetate extract with minimum inhibitory concentrations (MICs) of 500 mg/mL and lupeol 2 had MIC value of 500 mg/mL [62]. 6.5. Antidiarrhoeal Degu et al. [24] evaluated the antidiarrheal activities of chloroform and methanol leaf extracts of C. macrostachyus using the castor oil induced diarrheal model, charcoal meal test and antienteropooling test in mice. The test groups received various doses (300, 400, and 500 mg/kg and an additional dose of 1000 mg/kg for the aqueous fraction) of the fractions, whereas positive controls received either loperamide (3 mg/kg) or atropine (5 mg/kg) and negative controls received vehicle (10 ml/kg). In the castor oil induced model, the chloroform (at all test doses) and methanol (at 400 and 500 mg/kg) fractions delayed diarrheal onset and decreased stool frequency and weight of faeces. The chloroform and methanol fractions produced dose-dependent decline in the weight and volume of intestinal contents while the aqueous fraction did not have a significant effect [24]. These authors also found that all the fractions produced antimotility effect either at all doses (chloroform fraction) or at middle and higher doses (methanol and aqueous fractions). 6.6. Anticonvulsant and Sedative Bum et al. [50] evaluated anticonvulsant effects of crude extracts of C. macrostachyus using mice model (maximal electroshock (MES), strychnine (STR), pentylenetetrazol (PTZ), picrotoxin (PIC), isonicotinic hydrazide acid (INH))-induced convulsions and diazepam-induced sleep in assessing the sedative effects. Croton macrostachyus at the doses of 34 and 67 mg/kg protected 80, 80, 80, and 60% of mice from PIC, STR, PTZ, and MES-induced seizures, respectively [50]. Croton macrostachyus also delayed the onset to seizures in the INH test. The decoctions of C. macrostachyus possess sedative and anticonvulsant activities and these results corroborate the use of the species as herbal medicine for epilepsy in Ethiopia [36] and epilepsy and insomnia in Cameroon [50]. 6.7. Antidiabetic Arika et al. [88] evaluated in vivo hypoglycemic activity of aqueous leaf extracts of C. macrostachyus in male Swiss white albino mice. Aqueous leaf extract of C. macrostachyus was intraperitoneally and orally administered to alloxan (180.9 mg/kg; intraperitoneally) induced diabetic mice at different doses of 25 mg/kg body weight (bwt), 48.4 mg/kg bwt, 93.5 mg/kg bwt, 180.9 mg/kg bwt, and 350 mg/kg bwt and the effects on blood glucose levels investigated. Treatment of diabetic mice with doses of the leaf extract resulted in significantly lower levels of fasting blood glucose and the effects of the leaf extract were comparable with the conventional drugs [88]. Therefore, the results suggest that C. macrostachyus leaf extract is a potent hypoglycemic agent and this validates the use of root decoction as herbal medicine for diabetes in Tanzania [43]. 6.8. Anti-Inflammatory Kamanyi et al. [84] evaluated the antinociceptive and anti-inflammatory activities of the aqueous and methylene chloride/methanol stem bark extracts of C. macrostachyus. The extracts were administered orally at the doses of 150, 300, and 600 mg/kg and examined against pain induced by acetic acid, formalin, and pressure and against inflammation induced by carrageenan, histamine, and formalin. Results obtained by Kamanyi et al. [84] showed that both extracts induced dose-dependent reduction in the number of abdominal constrictions induced by acetic acid, and the three doses of the two extracts also reduced the two phases of pain induced by formalin. At the dose of 600 mg/kg, the aqueous and the methylene chloride/methanol extracts exhibited analgesic activity against pressure-induced pain. The two extracts also exhibited anti-inflammatory activity, the methylene chloride/methanol extract being the most active inhibited acute inflammation induced by carrageenan, histamine, and formalin, and both extracts reduced the chronic inflammation induced by formalin [84]. Nguelefack et al. [102] also evaluated the antinociceptive properties of the methanol/methylene chloride extracts of the stem bark of C. macrostachyus using mice models of persistent inflammatory and neuropathic pain and also assessed its mechanism of action. The methanol/methylene chloride extract was tested on Complete Freund Adjuvant- (CFA-) induced persistent thermal and mechanical pain, neuropathic pain induced by partial sciatic nerve ligation (PSNL), prostaglandin E2- (PGE2-) induced acute mechanical hyperalgesia, as well as on nociception induced by capsaicin in mice. Mechanical hyperalgesia was assessed using von Frey hair in awake mice. The mechanism of action of methanol/methylene chloride extract was evaluated by using glibenclamide on PGE2-induced hyperalgesia or rimonabant on capsaicin-induced pain [102]. The authors found that the methanol/methylene chloride extract administered orally at the doses of 250 and 500 mg/kg induced long lasting and significant antihyperalgesic effects on CFA-inflammatory and PSNL-induced neuropathic pain. The methanol/methylene chloride extract significantly reduced the mechanical hyperalgesia induced by PGE2 either when administered preventively or therapeutically [102]. The authors also found that the methanol/methylene chloride extract also significantly and time dependently inhibited the capsaicin-induced nociception. These studies show that C. macrostachyus extracts of the stem bark possess analgesic, anti-inflammatory, and antinociceptive properties corroborating the traditional use of the species in the treatment and management of different diseases and ailments, including pain and inflammation in tropical Africa. 6.9. Antileishmanial Gelaw et al. [94] evaluated the antileishmanial activities of a compound, crotepoxide 3 isolated from chloroform extracts of C. macrostachyus against promastigotes and amastigotes form of Leishmania aethiopica. The result of the study revealed that observed IC50 values of crotepoxide 3 to be 219.7 and 229.70 μg/ml against promastigotes and amastigotes, respectively, and therefore, less active when compared to the reference antileishmanial drugs amphotericin B and miltefosine with IC50 values of 0.03 and 0.12 μg/ml, respectively [94]. 6.10. Antioxidant Teugwa et al. [86] evaluated antioxidant activities of methanolic leaf extracts of C. macrostachyus using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging methods. Methanolic leaf extract of C. macrostachyus showed antioxidant activity with IC50 value of 0.11 mg/ml. The documented antioxidant activities of C. macrostachyus leaf extracts are probably due to flavonoids and phenols that have been isolated from fruits, leaves, and roots [24, 30, 47, 51, 55, 84–89, 93]. Flavonoids and phenolic compounds found in plants are known to have antioxidant properties [110]. 6.11. Antiplasmodial Owuor et al. [103] evaluated antiplasmodial activities of dichloromethane leaf and stem extracts of C. macrostachyus using the SYBR Green I fluorescence assay (MSF assay) with mefloquine and chloroquine as positive controls. The dichloromethane leaf and stem extracts were active against chloroquine sensitive Plasmodium falciparum strain with IC50 value of 2.720 ± 0.627 μg/ml [103]. Similarly, Bantie et al. [87] evaluated antiplasmodial activities of chloroform, methanol, and aqueous leaf extracts of C. macrostachyus using a rodent model of malaria. The rodent malaria parasite Plasmodium berghei was used to inoculate healthy male Swiss albino mice, 6–8 weeks old, and the parameters parasitemia, survival time, body weight, temperature, and packed cell volume were then determined using Peter's and Rane's tests [87]. Chemoprotective effect exerted by the extracts ranged between 12 and 91% and the chemotherapeutic effect of the extracts was in the range of 39–83%. The crude extracts prevented loss of weight and reduction in temperature but did not affect packed cell volume [87]. In another study, Mohammed et al. [104] evaluated antimalarial activities of the methanol and aqueous leaf extracts of C. macrostachyus using a 4-day suppressive standard test on Plasmodium berghei. Methanol and aqueous extracts of C. macrostachyus showed dose-dependent chemosuppressive effect at various doses in mice infected with Plasmodium berghei parasite while the crude methanol extracts of C. macrostachyus suppressed parasitaemia at all dose levels compared to the negative control groups but did not improve survival time [104]. The mice treated with the methanol and aqueous extracts at 600 mg/kg survived longer (10.60 ± 0.51 days for methanol extract and 9.60 ± 0.51 day for aqueous extract) than those in the negative control group with mean survival time of 6.2 ± 0.20 days [104]. The antimalarial activity test showed that C. macrostachyus exhibited significant antiplasmodial activity as evidenced by their ability to suppress Plasmodium berghei infection in mice in a dose-dependent manner, which may partly justify the claim by traditional practitioners about the use of these two plants against malaria. Mekonnen [89] also evaluated antiplasmodial activity of 80% methanol extract of the fruit and root of C. macrostachyus in a rodent model of malaria. The rodent malaria parasite Plasmodium berghei was used to inoculate healthy 8-week-old male Swiss albino mice and the parameters of parasitemia, survival time, body weight, temperature, and packed cell volume were determined using Peter's test and Rane's test [89]. Both extracts significantly inhibited parasitemia, increased survival time, prevented loss of weight and temperature, but did not affect the packed cell volume [89]. Results of this study suggest that the root and fruit extracts of C. macrostachyus have promising antiplasmodial activity against Plasmodium berghei in a dose-dependent manner, which supports the folkloric use of the plant for treating malaria. 6.12. Larvicidal Karunamoorthi and Ilango [19] evaluated larvicidal activities of methanol leaf extracts of C. macrostachyus against late third instar larvae of Anopheles arabiensis Patton, a potent malaria vector. The larval mortality was observed 24 h of posttreatment. The methanol leaf extracts showed different degree of mortality against the malaria vector Anopheles arabiensis with LC50 and LC90 values of 89.25 and 224.98 ppm, respectively. These results establish that C. macrostachyus could serve as potent mosquito larvicidal agent against Anopheles arabiensis although its mode of actions and larvicidal efficiency under the field conditions should be investigated and determined. 6.13. Toxicity The results of toxicity studies of aqueous and hydroalcoholic bark extracts of C. macrostachyus using albino mice showed LD50 value of 190.2 ± 15.7 mg/kg for aqueous extract and LD50 value of 87.5 ± 12.3 mg/kg for hydroalcoholic extract [73]. Desta [73] determined the median effective single dose of C. macrostachyus bark extract, that is, the dose that expelled Taenia saginata L. worms partially or totally in 50% of worm-infested human volunteers. Croton macrostachyus showed median effective single dose of 6.42 ± 0.82 and the number of hours that elapsed before partial or total expulsion of the worms following administration of C. macrostachyus was 12.9 ± 2.1 hours. Gadir et al. [106] evaluated oral toxicity of C. macrostachyus seeds in Nubian goat kids through clinical, hematological, and pathophysiological parameters. The Nubian goat kids were allotted as untreated controls and ground C. macrostachyus seeds were given to kids in repeated daily oral doses of 1 g/kg or 0.25 g/kg. Both oral dose levels of C. macrostachyus seeds were lethal for kids between days 7 and 21 and caused bloody diarrhoea, dyspnea, dehydration, loss in condition, paresis of the hind limbs, and recumbency before death [106]. Lesions in the affected animals included widespread hemorrhages and congestion, enterohepatonephrotoxicity, pulmonary hemorrhage, emphysema and cyanosis, tracheal froths, ascites, and hydropericardium. These lesions were accompanied by increases in the activity of serum AST, in the concentration of urea, and decreases in total protein and albumin, anemia, and leukopenia [106]. Moshi et al. [43] evaluated toxicity of root aqueous ethanol extracts of C. macrostachyus using the brine shrimp lethality test. Croton macrostachyus demonstrated moderate toxicity with concentration killing 50% (LC50) of the shrimps at 13.40 μg/ml. These findings indicate the possibility that C. macrostachyus extracts may be toxic or contain useful cytotoxic compounds, which were not reported by the traditional healers. Wagate et al. [96] evaluated cytotoxicity of leaf and root methanolic extracts of C. macrostachyus using the brine shrimp lethality test. Croton macrostachyus showed LC50 value of 387 μg/mL, which was considered to be relatively nontoxic [96]. Mbiantcha et al. [107] evaluated toxicity of aqueous and methylene chloride/methanol extracts of C. macrostachyus stem bark using Artemia spp. lethality assay, mice and Wistar rats. For the cytotoxicity study, the aqueous and organic extracts were administered to larvae of Artemia spp. and the number of deaths was determined after 6 hours and 24 hours, while for the acute study, the extracts were administered to mice. In the cytotoxicity study, aqueous and organic extracts showed LC50 values of 569 and 425 μg/ml, respectively. In acute toxicity study, aqueous extract did not provoke death until the dose 16 g/kg, whereas the organic extract caused general behaviors, adverse effects, and mortality. Mortality increased with increasing doses, with LD50 values of 10.2 and 9.4 g/kg bwt, respectively, for male and female mice [107]. In another acute toxicity study in mice, the methanol leaf extract of C. macrostachyus at a single oral doses of 2 and 5 g/kg bwt caused no mortality within the first 24 h and up to 14 days observation period [87]. Results from the study suggested safety profile of this herbal extract in the study mice. Physical and behavioral observations of the experimental mice did not show any visible signs of overt toxicity such as lacrimation, loss of appetite, tremors, hair erection, salivation, and diarrhoea. These studies revealed that C. macrostachyus is not toxic in both acute and subacute tests at the tested doses of the extracts. In an in vivo study, the methanol extract of C. macrostachyus showed dose-dependent chemosuppressive effect at various dose levels, that is, 200 (21.1%), 400 (27.7%), and 600 (34.3%) mg/kg body weight in Plasmodium berghei-infected mice [104]. The mice treated with chloroquine were completely free from parasitemia on day 4 in all groups (100% suppression). The crude methanol extract of C. macrostachyus significantly suppressed parasitemia at all dose levels compared to the negative control groups (distilled water) but did not significantly prolong the survival time of infected mice. Similarly, the aqueous extract at the doses of 200, 400, and 600 mg/kg body weight significantly reduced % parasitemia (26.14, 30.50, and 50.53%, resp.) compared to the negative control group in this study. Mbunde et al. [27] evaluated toxicity of leaf dichloromethane extracts of C. macrostachyus using the brine shrimp lethality test. Croton macrostachyus demonstrated moderate toxicity with concentration killing 50% (LC50) of the shrimps at 12.94 μg/ml [27]. Omosa et al. [108] also evaluated the cytotoxicity of dichloromethane and methanol (1 : 1) extract of C. macrostachyus stem bark using the resazurin reduction assay against CCRF-CEM leukemia cell line. The dichloromethane and methanol extract of C. macrostachyus stem bark displayed cytotoxicity towards leukemia CCRF-CEM cells with IC50 value of 60.6 μg/mL [108]. Based on the cytotoxicity studies done on C. macrostachyus crude extracts [27, 43, 73, 96, 104, 106–108], it can be concluded that caution must be exercised in the use of the species as herbal medicine. Go to: 7. Conclusion The present review summarizes the ethnomedicinal uses and recent findings on phytochemistry, pharmacology, and cytotoxicity of different extracts and compounds of C. macrostachyus. Alkaloids, amino acids, anthraquinones, carbohydrates, cardiac glycosides, coumarins, essential oil, fatty acids, flavonoids, phenolic compounds, phlobatannins, polyphenols, phytosteroides, saponins, sterols, tannins, terpenoids, unsaturated sterol, vitamin C, and withanoides have been demonstrated to be the main active ingredients of C. macrostachyus. Pharmacological studies have also focused on evaluating anthelmintic, antibacterial, antimycobacterial, antidiarrhoeal, antifungal, anticonvulsant and sedative, antidiabetic, anti-inflammatory, antileishmanial, antioxidant, antiplasmodial, larvicidal, and cytotoxicity activities of the different extracts and compounds isolated from C. macrostachyus. Future research should focus on the mechanisms of action of bioactive constituents of the species to illustrate the correlation between the ethnomedicinal uses and pharmacological properties of the species. Since previous studies have established that C. macrostachyus may contain potentially toxic compounds, there is need for detailed toxicological review of the crude extracts and pure compounds of the species. Lastly, since C. macrostachyus is widely used in combination with other plant species in various herbal concoctions, there is need for extensive research to evaluate synergistic effects of the different extracts or pure isolates to evaluate their ability to enhance the efficiency of the additive mixtures. Go to: Acknowledgments The author would like to express his gratitude to the National Research Foundation (NRF) and Govan Mbeki Research and Development Center (GMRDC), University of Fort Hare, for financial support to conduct this research. Go to: Conflicts of Interest The author declares that he has no conflicts of interest. Go to: References 1. Smith A. R. Euphorbaceae. In: Polhill R. M., editor. Flora of Tropical East Africa. Rotterdam, The Netherlands: A A Balkema; 1987. pp. 20–391. 2. Gilbert M. G. Flora of Ethiopia and Eritrea. In: Edwards S., Mesfin T., Hedberg I., editors. Euphorbiaceae. Addis Ababa, Ethiopia: Addis Ababa University; 1995. pp. 265–380. 3. 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Antioxidant effect of aqueous extract of four plants with therapeutic potential on gynecological diseases; Semen persicae, Leonurus cardiaca, Hedyotis diffusa, and Curcuma zedoaria

Eur J Med Res. 2017; 22: 50. Published online 2017 Nov 25. doi: 10.1186/s40001-017-0293-6 PMCID: PMC5702154 Shaojian Ji, Amir Fattahi, Nathalie Raffel, Inge Hoffmann, Matthias W. Beckmann, Ralf Dittrich,corresponding author and Michael Schrauder Author information ► Article notes ► Copyright and License information ► OB/GYN, University Hospital Erlangen, Friedrich-Alexander University, Universitätsstrasse 21-23, Erlangen-Nürnberg, 91054 Erlangen, Germany Shaojian Ji, Email: ed.liamtoh@naijoahsij. Contributor Information. corresponding authorCorresponding author. Abstract Background Little information is available concerning antioxidant effects of plant teas (water boiled) which are used more commonly in traditional Chinese medicine than other extracts. Thus, we addressed this issue by evaluating the ability of teas from four different plants with therapeutic potential on gynecological diseases. Methods The aqueous extracts of Semen persicae, Leonurus cardiaca, Hedyotis diffusa, and Curcuma zedoaria rhizome were prepared and then their effects on copper-induced low-density lipoprotein cholesterol (LDL-C) oxidation were evaluated by spectrophotometric method. Density gradient ultracentrifugation method was recruited to isolate LDL-C from healthy individuals. Results Our results showed that adding 10, 20, and 30 µl S. persicae could increase the lag phase duration of LDL-C oxidation compared with control reaction 12, 21, and 33%, respectively. The most effective delay (87%) was observed when 30 µl H. diffusa was added to the reaction. In cases of L. cardiaca and C. zedoaria, we found no significant influence on the lag phase duration (p > 0.05). Moreover, our findings about starting point of the decomposition phase were almost in parallel with the lag phase results, as 30 µl of S. persicae or H. diffusa teas could significantly increase the initiation time of decomposition (p < 0.05). Conclusions In conclusion our results showed that both S. persicae and H. diffusa teas and not L. cardiaca and C. zedoaria could have medicinal therapeutic effects partly through direct oxidation prevention. Keywords: Antioxidants, Curcuma zedoaria, Herbal medicine, Hedyotis diffusa, Leonurus cardiac, Semen persicae Go to: Background Oxidative stress, an imbalance between formation and elimination of reactive oxygen species (ROS), is one of the major causes of many diseases including cancer [1], diabetes mellitus [2], atherosclerosis [3], and more importantly gynecological diseases especially endometriosis [4], polycystic ovarian syndrome (PCOS) [5], and menstruation disturbances [6, 7]. Such negative effects of excessive ROS are due to oxidative damage of various cellular components including lipids, proteins, and nucleic acids [8]. Previous studies have mentioned the beneficial and preventive effects of antioxidant substances on oxidative stress-induced diseases via scavenging free radicals [9, 10]. In this concept, therapeutic effects of various antioxidants on female reproductive system diseases such as dysmenorrhea [11, 12], PCOS [13], and endometriosis [14] have been documented. Although, artificial antioxidants are extensively used in food products and medications but due to their instability and also possible potential in acting as carcinogens, there is a growing interest towards using natural antioxidants [15, 16]. In this case, herbal medicinal plants which are well-known in folk medicine and contain potent antioxidant substances such as phenolic acid, flavonoids, and tannins have attracted a considerable attention [17, 18]. Thus, numerous studies have been conducted to investigate therapeutic and preventive roles of various herbal extracts in gynecological diseases. For example, in a study conducted by Yang et al. [19] beneficial influence of Panax Ginseng Meyer on gynecologic complaints including menstrual irregularity and pain through improving oxidative status has been indicated. However, little information is available concerning antioxidant capacity of some herbal plants of which aqueous extractions are used as teas in traditional Chinese medicine; their medicinal potential in female health has been emphasized. The Semen persicae, also called Taoren (or peach kernel), consists of the substantial part of the harvested peaches which is mostly considered as waste and low-value residue [20]. However, the S. persicae is considered as one of the important herbal products in traditional Chinese medicines [21]. Its beneficial effects on various pathological situations has been indicated including diverse womb as well as abdominal disorders and most commonly blood stasis [22]. Besides, its lipid and Malondialdehyde (MDA) lowering properties have been documented [23]. Moreover, based on the ancient literature this plant could regulate menstruation and relieve pain (reviewed in Ref. [21]). However, based on our knowledge its possible antioxidant potency especially as aqueous extract has not been investigated yet. Leonurus cardiaca also commonly called motherwort can be found widely throughout Europe and Asia [24]. The L. cardiaca has a 100-year history of use for the treatment of nervous and functional cardiac disorders in Europe [25]. Moreover, for many years, traditional Chinese medicines have applied this plant to treat different disorders such as blood stasis, irregular menstrual cycle, urine excretion problems, and inflammatory diseases [26]. Besides, traditional application of this plant in menopause and menstrual disorders (dysmenorrhea or absent menstruation) has been reported [27, 28]. Further researches also confirmed its antinociceptive, hypoglycemic, anticancer, antibacterial, antifungal, and antioxidant properties [29, 30]. However, the antioxidant capacity of its tea has not been sufficiently addressed yet. Hedyotis diffusa Willd (Oldenlandia diffusa, Rubiaceae) is an ancient Chinese herbal which can be found in Asian countries including China; its aqueous extract (boiled in water) is employed to treat many kinds of diseases [31]. Applying of the H. diffusa for treatment of diverse types of inflammation and cancer have been reported previously [32]. Besides, investigations have shown antifungal, anti-inflammatory, immunoregulatory, and antioxidant attributes of this plant [33] that make this plant a potential therapeutic herbal for female-specific disorders; however, based on our knowledge there is no study about therapeutic application of H. diffusa in female reproductive system diseases. Huan-huan et al. [34] demonstrated that alcohol-ethyl acetate extract of this plant has radical scavenging activity and could protect DNA from hydroxyl radical-induced damages. However, antioxidant potency of the H. diffusa tea is still unknown. Curcuma zedoaria (Rosc. Zingiberaceae), a plant found in tropical countries, is widely used in the traditional Chinese medicine, especially its tea that is made from dried rhizomes [35]. This plant is clinically applied in treatment of cancers, stomach diseases, chronic pelvic inflammation, blood stagnation, coronary heart disease, and anemia [36]. Powder of the dried plant is also applied for treatment of menstrual irregularities [37]. Previous studies have confirmed its antimicrobial, anti-inflammatory, and analgesic activities [38]. More interestingly, there are evidences about effective antioxidant capacity of the C. zedoaria rhizomes essential oil as well as aqueous and methanolic extracts [38, 39]. Considering pivotal roles of oxidative stress in various disorders such as gynecological diseases there is wide interest to use herbal medicinal plants which contain antioxidant substances to prevent such oxidation. On the other hand, most studies have paid attention on oils or other extracts of the plants and little information is available concerning antioxidant effect of the plant teas (water boiled) which are used more commonly than other extracts. Thus, we addressed this issue by evaluating the ability of teas from four different plants to prevent copper-catalyzed low-density lipoprotein cholesterol (LDL-C) oxidation; in particular, we investigated antioxidant activity of four plants with therapeutic potential on gynecological diseases, S. persicae, L. cardiaca, H. diffusa, and C. zedoaria tea. Go to: Methods LDL-C fraction isolation, purification, and measurement Fasting blood samples were collected from healthy volunteers into EDTA-containing tubes and subsequent plasma separation was performed by centrifugation. LDL-C isolation was carried out immediately using density gradient ultracentrifugation. We applied the Abbey et al. [40] protocol briefly as follows: Firstly, using KBr, the plasma density was adjusted to 1.21 g/ml and then the plasma was layered under NaCl solution (d = 1.006 g/ml) containing 0.1% EDTA in Quickseal tubes. After preparation of the tubes, they were centrifuged at 4 °C and 280,000×g for 6 h by sequential ultracentrifugation (Beckman Ultracentrifuge, type L5-75, Ti 75 rotor). Afterwards, the LDL-C band (yellow band) was collected. In order to purify the LDL-C particles we applied the gel filtration method. Briefly, the gel bed column was washed twice with PBS buffer and then 800 μl of isolated LDL-C were added to the column; afterwards, the LDL-C was eluted from the column by adding 2.6 ml PBS buffer. To confirm purity of isolated fractions, agar-agarose gel electrophoresis was employed; the results showed that the LDL-C content was more than 96% in all eluates. LDL-C cholesterol levels were also evaluated in each eluate based on an enzymatic colorimetric method using automatic analyzer (Olympus AU 2700, NY, USA). Preparation of the teas Dried forms of S. persicae, L. cardiaca, H. diffusa, as well as C. zedoaria rhizome were purchased from a traditional Chinese pharmacy (Wenzhou, China). Considering that these herbal plants commonly are used as tea through boiling in water, we also prepared the teas with this method. For this purpose, 20 g of each dried plants were added into 100 ml distilled water and the water was heated for 20 to 30 min until it started to boil. After 20 min of boiling, the aqueous extracts were filtered to avoid any solid particles; we let the teas cool down to room temperature and used them freshly for antioxidant evaluation. Evaluation of LDL-c oxidation delay The copper-induced LDL-C oxidation is accompanied by an increase in levels of dienes which have a maximum absorbance at 234 nm. The LDL-C oxidation consists of three phases including lag, propagation, and decomposition phase [41]. The lag phase is an oxidation-resistant step in which diene levels and consequently absorbance at 234 nm is almost constant. However, upon the propagation phase, the absorbance quickly increases and finally reaches the maximum level. In the present study we evaluated direct effects of different tea concentrations on preventing LDL-C oxidation and delaying the lag phase duration. For this purpose, 10, 20, and 30 µl of each tea were added into a 0.08 mg LDL-C-containing eluate and the final reaction volume was adjusted to 1 ml using O2-saturated PBS buffer. Adding CuSO4 to the reaction and mixing the reaction batch initiated the oxidation process; absorbance at 234 nm was continuously measured at 30 °C for maximum 2 h (depending on the lag phase duration). Two reactions without tea (control) and two additional reactions for all three tea concentrations (10, 20, and 30 µl) were carried out for each tea simultaneously. Besides, all reactions were conducted three times. The intercept of the baseline and slope of the absorbance curve in the propagation phase was defined as the lag phase; the data were expressed in minutes. The peak time was considered as the time of maximum absorbance (Fig. 1). Fig. 1 Fig. 1 An example for kinetics of LDL oxidation in control and herbal tea added reactions Go to: Statistical analysis Kolmogorov–Smirnov test was used to confirm the normal distribution of data. One-Way ANOVA test following Tukey’s post hoc test was applied to compare the data among groups. All Statistical analyses were conducted using SPSS (version 16). Differences between means were considered significant when p value was 0.05 or less. Go to: Results The effects of S. persicae, L. cardiaca, H. diffusa, and C. zedoaria teas with various doses (0, 10, 20, and 30 µl) on duration of LDL-C oxidation lag phase are shown in Table 1. Our results showed that adding 30 µl S. persicae tea to the LDL-C oxidation reaction could significantly delay the lag phase in comparison to the control reaction without tea (p = 0.018). The lag phase durations in lower doses of S. persicae tea (10 and 20 µl) were also higher than the control (without tea); nevertheless, this difference did not reach statistical significance (p > 0.05). In case of H. diffusa tea, we also observed a dose-dependent correlation between tea amount and lag phase duration. As adding 20 µl of the tea to the reaction could cause 29.63 ± 2.12 min lag phase which was significantly higher than lag phase duration in reactions without or with 10 µl of the tea (p = 0.001 and 0.023, respectively). Moreover, we found that the lag phase duration in reactions with 30 µl H. diffusa tea was statistically significant longer than the reactions without or with 10 and 20 µl of the same tea (p < 0.001, p < 0.002 and p = 0.032, respectively). Table 1 Table 1 Effect of four herbal teas on the lag phase duration (min) of copper-induced low-density lipoprotein cholesterol oxidation In order to provide better understanding about effects of the teas on LDL-C oxidation we calculated the lag phase modification of each tea in comparison to corresponding reactions without the tea and illustrated the results as percentage in Fig. 2. We found that higher amounts of the teas (30 µl) could have a higher effect on duration of the lag phase as adding 10, 20, and 30 µl S. persicae could increase the phase duration compared with control reaction, respectively, 12, 21, and 33%. The most effective delay was observed when 30 µl H. diffusa was added to the reaction; it caused about 87% increase in the lag phase duration. Adding 10 and 20 µl of H. diffusa caused a lag phase extension of 22 and 55%, respectively. We further evaluated peak time in all reactions; peak time shows the starting point of the decomposition phase in LDL-C oxidation (Table 2). The obtained results were almost in parallel with our findings about lag phase, as addition of 30 µl S. persicae and H. diffusa tea could significantly increase the peak time compared to reactions without and with 10 µl of the teas. (for S. persicae p = 0.001 and 0.016 and for H. diffusa p = 0.003 and 0.009, respectively). Fig. 2 Fig. 2 Percentage of the lag phase duration modification in low-density lipoprotein cholesterol oxidation following addition of four teas. All three different quantities per tea are depicted and the control reaction is set as 100% Table 2 Table 2 Comparison of the peak time (min) of copper-induced low-density lipoprotein cholesterol oxidation among reactions with four herbal teas Go to: Discussion Considering existence of wide interest among people to use various medicinal plants as teas and also due to lack of information about antioxidant effects of such teas, we investigated antioxidant potency of S. persicae, L. cardiaca, H. diffusa, and C. zedoaria teas in preventing LDL-C oxidation in the present study. In case of S. persicae, we found that adding 30 µl of the tea to LDL-C oxidation reaction could prolong the lag phase and prevent oxidation of LDL-C. An ancillary increase in peak time of LDL-C oxidation following the tea adding demonstrated the potential of this tea in even decreasing oxidation rate; however, lack of significant effects with lower amounts (10 and 20 µl) revealed that the antioxidant capacity is not that much potent in these cases. Previous studies on S. persicae, have mostly focused on its anti-coagulatory and anti-inflammatory effects; although the latter effect could also be partly a result of its antioxidant activity [42]. However, in accordance with our findings, Wu et al. [43] have reported the ability of S. persicae meal in scavenging radicals, notwithstanding they used various chemical solutions such as petroleum ether to extract the meal. Moreover, in an in vivo study, it has been demonstrated that oral administration of S. persicae decoction in hyperlipidemia patients could decrease plasma levels of MDA which is a lipid peroxidation marker [44]. Considering the ability of the tea in delaying LDL-C lag phase as well as slowing oxidation propagation phase, it could be postulated that the antioxidant activity of S. persicae tea includes chelation of copper ions, neutralizing free radicals, and decomposing ox-LDL-C. Such antioxidant activities could be obtained from phenolic compounds of the tea [45]. Although we did not evaluate phenolic composition of the tea; nevertheless, previous studies showed the existence of high amounts of phenolic compounds in S. persicae meals extracted by Soxhlet extractor [43]. So most likely the tea also has such components and could play a role as an antioxidant solution. Higher antioxidant activity of 30 µl tea in comparison to lower amounts in preventing LDL-C oxidation is logical as the higher tea amount would have higher quantities of phenolic compounds and an increased antioxidant activity; this is in agreement with previously reported findings [43, 46]. So it could be postulated that therapeutic effect of S. persicae on endometriosis [47], removing blood stasis [22], and menstrual irregularity [21] could be partly through its antioxidant properties, as an association between oxidative stress and these disorders has been previously reported [4, 6, 7]. H. diffusa, like S. persicae, showed antioxidant activity even in lower amounts. We found that adding 30 µl of the H. diffusa tea to the LDL-C oxidation reaction could prolong the lag phase about 87% and also could significantly increase the peak time. Such a strong inhibitory effect of this tea in inhibiting LDL-C oxidation could introduce the H. diffusa tea as a potential drink to prevent oxidative stress-related gynecological diseases such as PCOS and endometriosis. However, further in vivo studies on patients with oxidative stress-related diseases are required to clarify the tea beneficial influences. Although the water extract of this plant has not been evaluated in terms of antioxidant activity and just its anticancer properties have been indicated [31], Bhuyan et al. [48] have reported radical scavenging capacity of the plant ethanol extract; this is in confirmation of our study. Besides, Huan-huan and colleagues [34] have documented that alcohol-ethyl acetate extract of H. diffusa has the ability to chelate ferrous iron, so one of the possible mechanisms of the tea in preventing copper-induced LDL-C oxidation might be chelating copper ions. Another possible reason for inhibitory effects of H. diffusa on LDL-C oxidation could be the presence of phenolic compounds such as phenolic acids and flavonols, as reported previously [49]. Moreover, in a study conducted by Yan et al. [50], four polysaccharides with strong hydroxyl radical scavenging activity in water/alkaline extraction and ethanol precipitation of H. diffusa have been detected. Yan et al. also claimed that higher doses from two of the polysaccharides have a more significant radical scavenging activity than vitamin C at the same dose. The presence of such polysaccharides in the tea could also be considered as a reason for its antioxidant capacity. Interestingly, we found no statistically significant effect of L. cardiaca tea on LDL-C oxidation. Contrary to our findings, antioxidant activity of L. cardiaca alcoholic extract has been reported in both in vitro and in vivo conditions [51, 52]. Lack of significant effects of L. cardiaca tea on LDL-C oxidation in our study could be considered from different points of view. First, it is possible that reported beneficial influences of L. cardiaca on oxidative stress and lipid peroxidation are results of indirect effects of the plant rather than the direct mechanism. Supporting this idea, it has been mentioned that the antioxidant effects of L. cardiaca are only present in oxidative stress conditions and most probably through increasing antioxidant enzyme activities such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) [53]. However, reports about in vitro antioxidant activity of the plant extract implies potential of a direct radical scavenging ability [30]. Second explanation for our contrary results could be absence or low concentration of antioxidant compounds in L. cardiaca plant; however, reports about presence of antioxidant compounds such as flavonoids, triterpenoids, and alkaloids in the plant extracts rejects such a hypothesis [54]. Another possible reason could be inadequacy of water as a solvent for preparing L. cardiaca extract with antioxidant capacity. In support of this reason, it has been reported that the water extract of L. cardiaca fruits had lower antioxidant activity compared with ethyl acetate, propyl alcohol, and supercritical CO2 extracts [55]. Thus, adding higher amounts of water-boiled extract (tea) might have caused a statistically significant effect on LDL-C oxidation. However, it should be mentioned that our aim was to evaluate antioxidant capacity of herbal medicine prepared using traditional Chinese medicine method rather than lipophilic extraction methods. Another possible reason could be inadequacy of water as a solvent for preparing L. cardiaca extract with antioxidant capacity. In support of this reason, it has been reported that the water extract of L. cardiaca fruits had lower antioxidant activity compared with ethyl acetate, propyl alcohol, and supercritical CO2 extracts [55]. Thus, adding higher amounts of water-boiled extract (tea) might have caused a statistically significant effect on LDL oxidation. A further potential reason for lack of antioxidant capacity in this plant could be due to the thermal condition in the tea preparation. However, this possibility seems unlikely because the plant was boiling for up to 20 min and the time duration is not comparable with the studies that have reported adverse effects of heating [56]. On the other hand, some studies even reported beneficial effect of such a short heating for increasing antioxidant activity of the herbal extracts [57, 58]. Like L. cardiaca tea, the tea prepared from C. zedoaria also did not have a significant influence on LDL-C oxidation which is in contradiction with previous findings. Previous studies have indicated antioxidant activity of C. zedoaria essential oil [38]. Also it has been reported that C. zedoaria extracts, prepared using methanol, chloroform, hexane, and ethyl acetate, could have various antioxidant activities such as scavenging and chelating abilities [39, 59]. The abovementioned reasons for L. cardiaca could also be considered as explanations for contradictory results in case of L. cardiaca tea. Go to: Conclusions In conclusion, our results show that both S. persicae and H. diffusa teas could prevent and also decrease the rate of LDL-C oxidation. However, statistically significant effects were not found for the L. cardiaca and C. zedoaria teas, possibly due to an inefficient extracting method and/or low antioxidant capacity of the plants. Considering the high prevalence of consuming medicinal plants as teas and on the other hand necessity of evaluating potential antioxidant capacity as well as side effects of such teas, future studies should shed more light on these issues; including beneficial effects in oxidative stress-related gynecological diseases. Go to: Authors’ contributions SJ: laboratory analysis and experimental works and acquisition of data. AF: statistical analysis and manuscript drafting. NR, LL, IH: laboratory analysis and experimental works. MB: manuscript critical revision. RD: study design, interpretation of data, and revision of the manuscript. MS: manuscript critical revision and statistical analysis. All authors read and approved the final manuscript. Acknowledgements The research for this study was performed by Shaojian Ji in fulfillment of the requirements for the M.D. degree at Friedrich-Alexander University, Erlangen–Nürnberg, Germany. Competing interests The authors declare that they have no competing interests. Availability of data and materials Data and material are available upon request. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Funding No specific funding was received for the study. This study was financially supported by institutional funding. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Go to: Abbreviations MDA malondialdehyde PCOS polycystic ovarian syndrome ROS reactive oxygen species SOD superoxide dismutase GPx glutathione peroxidase Go to: Contributor Information Shaojian Ji, Email: ed.liamtoh@naijoahsij. Amir Fattahi, Email: ed.negnalre-ku@ihattaf.rima. Nathalie Raffel, Email: ed.negnalre-ku@leffaR.eilahtaN. Inge Hoffmann, Email: ed.negnalre-ku@nnamffoh.egni. Matthias W. Beckmann, Email: ed.negnalre-ku@nnamkceb.saihttam. Ralf Dittrich, Phone: +49 (0) 9131 8533553, Email: ed.negnalre-ku@hcirttid.flar. Michael Schrauder, Email: ed.negnalre-ku@nretxe.reduarhcs.leahcim. Go to: References 1. Gill JG, Piskounova E, Morrison SJ. Cancer, oxidative stress, and metastasis. In: Cold Spring Harb Symp Quant Biol. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2016. [PubMed] 2. Asmat U, Abad K, Ismail K. Diabetes mellitus and oxidative stress—a concise review. Saudi Pharm J. 2016;24:547–553. doi: 10.1016/j.jsps.2015.03.013. 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Rapidly changing climatic conditions for wine grape growing in the Okanagan Valley region of British Columbia, Canada

Sci Total Environ. 2016 Jun 15;556:169-78. doi: 10.1016/j.scitotenv.2016.02.200. Epub 2016 Mar 11. . Rayne S1, Forest K2. Author information 1 Chemologica Research, 1617-11th Avenue NW, Moose Jaw, Saskatchewan S6H 6M5, Canada. Electronic address: sierra.rayne@alumni.ubc.ca. 2 Department of Environmental Engineering Technology, Saskatchewan Polytechnic, 600 Saskatchewan Street West, Moose Jaw, Saskatchewan S6H 4R4, Canada. Abstract A statistical analysis was conducted on long-term climate records for sites bordering Okanagan Lake in the Okanagan Valley viticultural region of British Columbia, Canada. Average wine grape growing season temperatures are increasing rapidly in the area over the post-1980 period at rates upwards of 7.0±1.3°C/century. Similar increases in the average dormant season temperature are evident. These temperature changes are likely some of the most extreme observed among the world's wine producing areas during the past few decades. Growing degree day base 10°C (GDD10) has increased by nearly 50% at some locations since the 1970s, resulting in major impacts on the corresponding climate classification for viticulture. If current climate trends continue, the southern and central portions of the region will likely enter Winkler region II within the next few decades, placing them in the same category as well-established warmer wine regions from France, Spain, Italy, and Australia. The large dormant season temperature increases over the last several decades have resulted in the area no longer being a cold season outlier when compared to most other cool-climate viticultural areas. Based on average growing season temperatures, the southern end of Okanagan Lake has moved out of the cool-climate viticultural classification and into the intermediate zone, while the central and northern regions are now at the cool/intermediate viticulture interface, similar to the historical positions of the Rhine Valley in Germany, northern Oregon in the United States, and the Loire Valley, Burgundy-Cote, Burgundy-Beaujolais, and Champagne appelations of France. The corresponding suitable grape species for the area have evolved into warmer region varietals during this time frame, having substantial economic impacts on producers. Increased temperatures are also expected to bring greater threats from agricultural pests, notably Pierce's disease from the bacterium Xylella fastidiosa. KEYWORDS: Agriculture; Climate change; Grape growing; Viticulture; Wine production PMID: 26971218 DOI: 10.1016/j.scitotenv.2016.02.200

Effect of grape juice press fractioning on polysaccharide and oligosaccharide compositions of Pinot meunier and Chardonnay Champagne base wines.

Food Chem. 2017 Oct 1;232:49-59. doi: 10.1016/j.foodchem.2017.03.032. Epub 2017 Mar 8. Jégou S1, Hoang DA2, Salmon T2, Williams P3, Oluwa S2, Vrigneau C4, Doco T3, Marchal R2. Author information 1 Laboratoire d'Œnologie et Chimie Appliquée, URVVC EA 4707, Université de Reims Champagne-Ardenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France. Electronic address: sandrine.jegou@univ-reims.fr. 2 Laboratoire d'Œnologie et Chimie Appliquée, URVVC EA 4707, Université de Reims Champagne-Ardenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France. 3 INRA, UMR n°1083, Sciences Pour l'Œnologie, 2 Place Pierre Viala, 34060 Montpellier, France. 4 Institut Œnologique de Champagne, 9 Rue du Commerce, 51350 Cormontreuil, France. Abstract Press fractioning is an important step in the production of sparkling base wines to segregate the grape juices with different qualities. Grape juice fractions were collected during the pressing cycle at industrial and laboratory scales. The Pinot meunier and Chardonnay Champagne base wines obtained from the free-run juice and the squeezed juices exhibited strong differences from the beginning to the last step of pressing cycle for numerous enological parameters. Significant changes in polysaccharide (PS) and oligosaccharide (OS) base wine composition and concentration were found as the pressing cycle progressed. During the pressing cycle, the total PS concentration decreased by 31% (from 244 to 167mg/L) and 32% (from 201 to 136mg/L) in the Pinot meunier and Chardonnay wines respectively. The wine OS amounts varied between 97 and 139mg/L. The polysaccharide rich in arabinose and galactose (39-54%) and mannoproteins (38-55%) were the major PS in the base wines. KEYWORDS: Champagne wine; Chardonnay; Grape juice; Oligosaccharides; Pinot meunier; Polysaccharides; Press fractioning PMID: 28490102 DOI: 10.1016/j.foodchem.2017.03.032

Happy 2018

2017 - the year of my age

2017 was not a bad year for me, I have had much worse years. But I think it would all have been about my personal life/age if it was not for Editor-in-Chief: Barney Warf accepting my paper on the Cocoa Panyols of Trinidad over the objections of the reviewers. https://link.springer.com/article/10.1007/s10708-017-9835-2 I have blogged about my Cocoa Panyols paper before. http://tryl2012.blogspot.ca/2017/02/all-skin-teeth-eh-laugh.html Editor in Chief Warf found one or more of the same reviewers who rejected it from the Journal of Ethnopharmacology special issue on the Centre of the Americas, but he accepted my point of view that they were self-serving. Maybe my "academic age" helped me publish the paper without making any changes to please self-serving reviewers. Maybe the paper was about Geography all along. I should thank my secondary school Geography teacher for telling me to stop talking in his class because I needed a 3rd subject (Geography) for A levels since I was not going to pass Physics. I have to thank WUR MSc student Madeline Donald for unintentionally pushing me to publish the paper which I had decided to let sit until I could do more research on whether slaves could have possibly carried plants from Africa to the Americas. Madeline contacted me in July and arranged to visit me (a very enjoyable visit) because she was going to study "the useful plants being grown within the biodiverse “shade” of the Trinidadian cocoa plantations". I became suspicious at once and instead of being able to go back to the personal life I had no time for because I was submitting yet another grant application (http://tryl2012.blogspot.ca/2017/09/acknowledging-my-summer.html), I had to find a non-ethnobotany journal to submit my paper to, because I wanted my paper to be published before she finished her thesis. I also have to thank Rogério Miguel Puga, Professor Auxiliar at NOVA/FCSH for reading or having someone read part of my Harry Potter paper at the Harry Potter 20. International One-Day Conference in Portugal on Oct 20. Unfortunately the Editor of History & Theory, did not accept the longer version of the Harry Potter paper, after providing a more of less polite review, except for the non-apology for taking from April to December to review it and commenting that I did not know anything about the Philosophy of History. So I guess my readings on the subject were not apparent or maybe I am just an outsider. Of course all of my work would have been much harder if it were not for James K., acknowledging my temporary attachment to his workplace and providing (hopefully for keeps) my access to Microsoft Office... http://tryl2012.blogspot.ca/2013/08/microsoft-control.html href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3V-hgQ6NB8zzcu8J9jkxy7FtHGuNoEfXTpaBT8pSVrd8vhnO7iq4WphephnaKAdc7Az7HOcCAf06xvFOZ-jTuZqDTocShLTCtLvlqQZeYNUrx9-oZzOznNr19U0SzqFBNEKrAxUjfiQc/s1600/IMG_1559.JPG" imageanchor="1" >