Aqueous distillate of mature leaves of Vernonia zeylanica (L.) Less. and Mallotus repandus (Rottler) Müll. Arg. cued from traditional medicine exhibits rapid wound healing properties.

ETHNOPHARMACOLOGICAL RELEVANCE: Sri Lankan traditional medicine uses Vernonia zeylanica and Mallotus repandus broadly for the treatment of a multitude of disease conditions, including wound healing. AIM OF THE STUDY: We aimed to scientifically validate the safety and efficacy of wound healing of an aqueous distillate of Vernonia zeylanica and Mallotus repandus (ADVM) mature leaves, tested on primary human dermal fibroblasts. MATERIALS AND METHODS: Human dermal fibroblasts isolated from clinical waste from circumcision surgery were characterized by flowcytometry and trilineage differentiation. The MTT dye reduction assay, and the ex vivo wound healing scratch assay established wound healing properties of ADVM using the primary human dermal fibroblast cell line. Upregulation of genes associated with wound healing (MMP3, COL3A1, TGFB1, FGF2) were confirmed by RT qPCR. GC-MS chromatography evaluated the phytochemical composition of ADVM. RESULTS: Compared to the synthetic stimulant, ß fibroblast growth factor, ADVM at 0.25% concentration on the primary dermal fibroblast cell line exhibited significant ex vivo, (i) 1.7-fold % cell viability (178.7% vs 304.3 %, p < 0.001), (ii) twofold greater % wound closure (%WC) potential (47.74% vs 80.11%, p < 0.001), and (iii) higher rate of % WC (3.251 vs 3.456 % WC/h, p < 0.05), sans cyto-genotoxicity. Up regulated expression of FGF2, TGFB1, COL3A1 and MMP3, genes associated with wound healing, confirmed effective stimulation of pathways of the three overlapping phases of wound healing (P < 0.05). GC-MS profile of ADVM characterized four methyl esters, which may be posited as wound healing phytochemicals. CONCLUSIONS: Exceeding traditional medicine claims, the exvivo demonstration of rapid skin regeneration, reiterated by upregulated expression of genes related to wound healing pathways, sans cytotoxicity, propounds ADVM, cued from traditional medicine, as a potential safe and effective natural stimulant for rapid wound-healing. Additionally, it may serve as an effective proliferative stimulant of dermal fibroblasts for cell therapy, with potential in reparative and regenerative therapy of skin disorders.

Platelet augmentation activity of mature leaf juice of Sri Lankan wild type cultivar of Carica papaya L: Insights into potential cellular mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Carica papaya L., a common fruit crop of the family Caricaceae and its leaf juice/extract is a traditionally commended preparation against dengue and other thrombocytopenic diseases by many Asian countries. AIM OF THE STUDY: The present study posits the potential cellular mechanisms of platelet augmentation activity of mature leaf juice of Sri Lankan wild-type Carica papaya. MATERIALS AND METHODS: C. papaya leaf juice prepared from different cultivar types, maturity of the leaf, agro-climatic region, and preparation methods were orally administered to hydroxyurea-induced thrombocytopenic rats at 0.72 ml/100 g BW dosage to investigate the most potent platelet increasing preparation. The papaya juice doses; low dose (LD-0.18 ml/100 g BW), human equivalent dose (HED-0.36 ml/100 g BW), and high dose (HD-0.72 ml/100 g BW), were administered to thrombocytopenic rats (N = 6/group) daily for three consecutive days and post-treatment plasma levels of interleukin 6 (IL-6), thrombopoietin (TPO), and platelet-activating factor (PAF) were quantified using specific rat ELISA kits. The mature leaf juice of C. papaya induced IL-6 secretion from bone marrow cell (BMC) cultures was quantified using ELISA. The ability of papaya juice to protect the platelet membrane, from the damage caused by the lytic agent was analyzed in vitro using the lactate dehydrogenase (LDH) assay. The effect of the mature leaf juice of C. papaya on secondary hemostasis was investigated using blood coagulation and clot hydrolyzing activity. RESULTS: The comparative analysis revealed that the platelet increasing activity of C. papaya leaf did not significantly differ among different types of cultivar, maturity of the leaf, agro-climatic regions and preparation methods (p > 0.05). Both TPO and PAF levels in thrombocytopenic rats diminished when treated with all three doses of the mature leaf juice of C. papaya (p < 0.05), yet IL-6 plasma level was unaltered (p > 0.05). Nevertheless, ex vivo treatment of the mature leaf juice of C. papaya had significantly enhanced IL-6 levels of rat BMC cultures (p < 0.05). Pre-treatment of platelets with the mature leaf juice of C. papaya at different concentrations significantly inhibited LDH leakage from platelets and may have reduced the membrane damage caused by the lytic agent (p < 0.05). Treatment of mature leaf juice of C. papaya also significantly reduced blood clotting time through the extrinsic pathway of the blood coagulation cascade (p < 0.05). Further, prolonged incubation of the plasma clot with different concentrations of the papaya leaf juice revealed dose-dependent hydrolysis of the blood clot, indicating fibrinolysis activity. CONCLUSIONS: The current study exceeded the traditional medicinal claims, and scientifically affirmed the platelet augmentation activity of mature leaf juice of C. papaya. The mechanistic rationale tested herein explicated that the platelet augmentation activity of the papaya leaf juice can be partially attributed to the stimulation of bone marrow megakaryocytes via modulating thrombopoietic cytokines TPO and IL-6, and by inhibiting the secretion of PAF, while reducing the peripheral platelet destruction by stabilizing the platelet membrane. Further, mature leaf juice of C. papaya imparted both pro-coagulation and fibrinolysis activity of secondary hemostasis endorsing its potential against thrombocytopenia.

Therapeutic Potential of Skin Stem Cells and Cells of Skin Origin: Effects of Botanical Drugs Derived from Traditional Medicine.

Skin, the largest organ of the body, plays a vital role in protecting inner organs. Skin stem cells (SSCs) comprise a group of cells responsible for multiplication and replacement of damaged and non-functional skin cells; thereby help maintain homeostasis of skin functions. SSCs and differentiated cells of the skin such as melanocytes and keratinocytes, have a plethora of applications in regenerative medicine. However, as SSCs reside in small populations in specific niches in the skin, use of external stimulants for cell proliferation in vitro and in vivo is vital. Synthetic and recombinant stimulants though available, pose many challenges due to their exorbitant prices, toxicity issues and side effects. Alternatively, time tested traditional medicine preparations such as polyherbal formulations are widely tested as effective natural stimulants, to mainly stimulate proliferation, and melanogenesis/prevention of melanogenesis of both SSCs and cells of skin origin. Complex, multiple targets, synergistic bioactivities of the phytochemical constituents of herbal preparations amply justify these as natural stimulants. The use of these formulations in clinical applications such as in skin regeneration for burn wounds, wound healing acceleration, enhancement or decrease of melanin pigmentations will be in great demand. Although much multidisciplinary research is being conducted on the use of herbal formulas as stem cell stimulants, very few related clinical trials are yet registered with the NIH clinical trial registry. Therefore, identification/ discovery, in depth investigations culminating in clinical trials, as well as standardization and commercialization of such natural stimulants must be promoted, ensuring the sustainable use of medicinal plants.

Cytogenotoxicity evaluation of a heavy metal mixture, detected in a polluted urban wetland: Micronucleus and comet induction in the Indian green frog (Euphlyctis hexadactylus) erythrocytes and the Allium cepa bioassay.

Heavy metal contamination in wetland ecosystems is a serious environmental and health concern. This study evaluated the cytogenotoxicity of a previously evidenced heavy metal contamination (Cd, Cr, Cu, Pb and Zn ∼5 ppm each) in a polluted urban wetland, the Bellanwila-Attidiya sanctuary (BAS) in Sri Lanka, using a battery of cytogenotoxic assays. Micronucleus and comet assays evaluated the genotoxicity in erythrocytes of a common amphibian, the Indian green frog (Euphlyctis hexadactylus), under natural metal exposure in the wetland, and in vitro exposure, respectively.The Allium cepa bioassay assessed the cytogenotoxicity of the heavy metal mixture and of the individual metals, under laboratory exposure. Although in vivo natural exposure showed no significant induction of micronuclei in frog erythrocytes (P > 0.1), a significant and dose dependent elevation of comets was evident with in vitro exposure to the metal mixture (P < 0.001). Field controls did not show significant impacts in the A. cepa bioassay, whereas individual exposure to heavy metals reported lower effects than their combined exposure under laboratory conditions; Pb2+was the most toxic metal, with the highest mitotic inhibition (Pb2+>Cd2+>Zn2+>Cr6 >Cu2+), mutagenic potential as evaluated in the percentage incidence of chromosomal aberrations (Pb2+> Zn2+> Cu2+> Cr6+> Cd2+) and cytotoxicity evaluated by the incidence of cell apoptosis and necrosis (Pb2+>Cr6+>Cu2+>Cd2+>Zn2+). Thus, the test battery of micronucleus, comet and A. cepa assays that reveal differential aspects of cytogenotoxicity may serve as a valuable tool in environmental monitoring, primarily to screen for complex environmental mixtures of heavy metals that may impact ecological health.

Mature leaf concentrate of Sri Lankan wild type Carica papaya Linn. modulates nonfunctional and functional immune responses of rats.

BACKGROUND: The leaf concentrate of Carica papaya is a traditionally acclaimed immunomodulatory remedy against numerous diseases; nonetheless comprehensive scientific validation of this claim is limited. The present study thus investigated the immunomodulatory potential of Carica papaya mature leaf concentrate (MLCC) of the Sri Lankan wild type cultivar using nonfunctional and functional immunological assays. METHODS: Wistar rats (N = 6/ group) were orally gavaged with 3 doses (0.18, 0.36 and 0.72 ml/100g body weight) of the MLCC once daily for 3 consecutive days. Selected nonfunctional (enumeration of immune cells and cytokine levels) and functional (cell proliferation and phagocytic activity) immunological parameters, and acute toxic effects were determined using standard methods. Effect of the MLCC (31.25, 62.5, 125, 250, 500 and 1000 µg/ml) on ex vivo proliferation of bone marrow cells (BMC) and splenocytes (SC), and in vitro phagocytic activity of peritoneal macrophages (PMs), and their corresponding cytokine responses were evaluated. The phytochemical profile of the MLCC was established using liquid chromatography-mass spectrometry (LS-MS) and Gas chromatography-mass spectrometry (GC-MS). RESULTS: Counts of rat platelets, total leukocytes, lymphocyte and monocyte sub populations, and BMCs were significantly augmented by oral gavage of the MLCC (p < 0.05). The highest MLCC dose tested herein significantly reduced pro inflammatory cytokines, Interleukin 6 (IL-6) and Tumor Necrosis Factor α (TNF α) levels of rats (p < 0.05). The in vivo phagocytic index of rat PMs significantly increased by oral gavage of all three doses of the MLCC (p < 0.05). In vitro phagocytic activity of rat PMs were enhanced by the MLCC and triggered a Th1 biased cytokine response. The MLCC at low concentrations elicited ex vivo proliferation of BMC (31.25 µg/ml) and SC (31.25 and 62.5 µg/ml) respectively. Conversely, high concentrations (500 and 1000 µg/ml) exhibited cytotoxicity of both BMC and SC with significant modulation of cytokines. Chemical profile of the MLCC revealed the presence of several immunomodulatory compounds. The oral gavage of the MLCC was found to be safe in terms of both hepatic and renal toxicities. CONCLUSION: The present study established that the mature leaf concentrate (MLCC) of Carica papaya Sri Lankan wild type cultivar is orally active, safe and effectively modulates nonfunctional and functional immunological parameters of rats that unequivocally corroborate the traditional medical claims.

Potential role of herbal remedies in stem cell therapy: proliferation and differentiation of human mesenchymal stromal cells.

Stem cell therapy has revolutionized modern clinical therapy with the potential of stem cells to differentiate into many different cell types which may help to replace different cell lines of an organism. Innumerous trials are carried out to merge new scientific knowledge and techniques with traditional herbal extracts that may result in less toxic, affordable, and highly available natural alternative therapeutics. Currently, mesenchyamal stromal cell (MSC) lines are treated with individual and mixtures of crude herbal extracts, as well as with purified compounds from herbal extracts, to investigate the mechanisms and effects of these on stem cell growth and differentiation. Human MSCs (hMSCs) possess multilineage, i.e., osteogenic, neurogenic, adipogenic, chondrogenic, and myogenic, differentiation abilities. The proliferative and differentiation properties of hMSCs treated with herbal extracts have shown promise in diseases such as osteoporosis, neurodegenerative disorders, and other tissue degenerative disorders. Well characterized herbal extracts that result in increased rates of tissue regeneration may be used in both stem cell therapy and tissue engineering for replacement therapy, where the use of scaffolds and vesicles with enhanced attaching and proliferative properties could be highly advantageous in the latter. Although the clinical application of herbal extracts is still in progress due to the variability and complexity of bioactive constituents, standardized herbal preparations will strengthen their application in the clinical context. We have critically reviewed the proliferative and differentiation effects of individual herbal extracts on hMSCs mainly derived from bone marrow and elaborated on the plausible underlying mechanisms of action. To be fruitfully used in reparative and regenerative therapy, future directions in this area of study should (i) make use of hMSCs derived from different non-traditional sources, including medical waste material (umbilical cord, Wharton's jelly, and placenta), (ii) take account of the vast numbers of herbal extracts used in traditional medicine globally, and (iii) investigate the mechanisms and pathways of their effects on hMSCs.

Artemisia vulgaris L. ethanolic leaf extract reverses thrombocytopenia/thrombocytosis and averts end-stage disease of experimental severe Plasmodium berghei murine malaria.

BACKGROUND & OBJECTIVES: Artemisinin isolated from Artemisia annua is the most potent antimalarial against chloroquine resistant Plasmodium falciparum malaria. We previously reported that the ethanolic leaf extract of Artemisia vulgaris, an invasive weed and the only Artemisia species in Sri Lanka, possess both potent and safe antimalarial activity (in terms of antiparasitic properties) in a P. berghei murine malaria model. We report here a prototype study that investigated antidisease activities of A. vulgaris ethanolic leaf extract (AVELE) in a P. berghei ANKA murine malaria model that elicit pathogenesis similar to falciparum malaria. Profound thrombocytosis and thrombocytopenia in mice were detected in early-stage (Day 3), and at a later stage of infection (Day 6), respectively. Plasmodium berghei infected mice, 7 or 8 days post-infection reached end-stage disease with rapid drop in body temperature and usually die within 24 h, as a consequence of cerebral malaria. METHODS: Three doses of the AVELE (500, 750 and 1000 mg/kg) were used to assess antidisease activity of A. vulgaris in terms of survival, effects on thrombocyte related pathology and end-stage disease, antipyretic activity, and antinociception, using standard methodology. RESULTS: The 1000 mg/kg dose of AVELE significantly increased survival, reversed the profound thrombocytopenia/ thrombocytosis (p ≤0.01), altered the end-stage disease (p ≤0.05), and manifested significant antipyretic and antinociceptive (p ≤0.05) activities. INTERPRETATION & CONCLUSION: We conclude that a crude ethanolic leaf extract of A. vulgaris, showed potent antimalarial properties, in terms of antidisease activities; antipyretic activity, peripheral and central antinociception, increased survival, averted end-stage disease and reversed thrombocytopenia/thrombocytosis.

Antimalarial properties of Artemisia vulgaris L. ethanolic leaf extract in a Plasmodium berghei murine malaria model.

BACKGROUND & OBJECTIVES: Artemisinin isolated from Artemisia annua is the most potent antimalarial drug against chloroquine-resistant Plasmodium falciparum malaria. Artemisia vulgaris, an invasive weed, is the only Artemisia species available in Sri Lanka. A pilot study was undertaken to investigate the antiparasitic activity of an A. vulgaris ethanolic leaf extract (AVELE) in a P. berghei ANKA murine malaria model that elicits pathogenesis similar to falciparum malaria. METHODS: A 4-day suppressive and the curative assays determined the antiparasitic activity of AVELE using four doses (250, 500, 750 and 1000 mg/kg), Coartem® as the positive control and 5% ethanol as the negative control in male ICR mice infected with P. berghei. RESULTS: The 500, 750 and 1000 mg/kg doses of AVELE significantly (p ≤ 0.01) inhibited parasitaemia by 79.3, 79.6 and 87.3% respectively, in the 4-day suppressive assay, but not in the curative assay. Chronic administration of the high dose of AVELE ruled out overt signs of toxicity and stress as well as hepatotoxicity, renotoxicity and haematotoxicity. INTERPRETATION & CONCLUSION: The oral administration of a crude ethonolic leaf extract of A. vulgaris is non-toxic and possesses potent antimalarial properties in terms of antiparasitic activity.