SUMMARY AND CONCLUSION

Transcription

1 Chapter-7 SUMMARY AND CONCLUSION Introduction The history of traditional herbal drug or natural drug treatments had been reported in the Rigveda. The application of modem scientific techniques in natural drug treatments is an indicative of majority of world population rely on natural or herbal healthcare practices. In the ancient literature number of herbs had been reported for the hair growth promoting effect and to treat hair diseases like alopecia, dandruff, male and female sample baldness, hirsutism and so forth. Out of several plants based drugs, Bacopa monnieri, Emblica officinalis & Cyperus rotundus have been selected for the current research work for their hair growth promoting activity. Most often poly herbal formulations were reported for having promising hair growth promoting activities. Nevertheless the efficacy data has been confined due to non-existence of validated models and limited research work done by the researchers and other scientists. Usually the disease pertaining to hair loss is termed as alopecia, which is very common consequence of changes in hair cycle. There exists a major lacuna in availability of medicines for the treatment of alopecia. Only two synthetic drugs have been accredited by FDA, to begin with oral Finasteride and topical Minoxidil. However, their efficacy is confined due to the report associated with primary side effects. As a consequence, there's a demand and need to investigate new molecules from natural origin for stimulating hair growth potential, which should act as an alternative for the available synthetic molecules and which would be safe and effective for the said purpose. The present work addresses a challenge facing with the research and investigation of such molecule, which would be effective and safe and have minimum untoward effect to the patient in comparison to the available synthetic drugs. The main target of the present work was to compare and evaluate the effect of selected plant extracts and their 179

2 formulations on hair growth stimulating activities. The proliferative activities of the plant extracts evaluated utilizing in-vitro and in-vivo models, emphasizes that in preclinical reviews the ethanolic extracts of Bacopa monnieri, Emblica officinalis & Cyperus rotundus and the volatile oil extracted from Cyperus rotundus rhizome showed maximal hair growth stimulating effect amongst the various other plant extracts validated. For topical administration, it was preferred that the hair growth stimulating composition should have been formulated in an alcoholic extract, which could interfere in the absorption of the active ingredients present in the extract. Penetration enhancer like dimethyl sulphoxide (DMSO), propylene glycol and glycerol had been utilized in the formulations and preparations. The type of formulation and the quantity to be utilized by topical application was determined to a massive extent by the viscosity and surface area of application. Medicinal plants selected for the present work As per the plan, main plants selected for present work were Bacopa monnieri, Emblica officinalis & Cyperus rotundus. Emblica officinalis or Indian goose berry is one of most important drug in Indian traditional system, especially in Ayurveda. It belongs to family Euphorbiaceae. Dried fruits, fresh fruits, seed, leaves, root bark and flowers of Emblica officinalis (Amla) are widely used in medicines. The fruit is rich in quercetin, phyllaemblic compounds, gallic acid, tannins, flavonoids, pectin, and vitamin C and also contains various polyphenolic compounds. Bacopa monnieri (Brahmi), belonging to family Scrophulariaceae, has been used by Ayurvedic medical practitioners in India for almost 3000 years. It is a succulent creeping marshy herb with numerous branches, small oblong leaves & rooting at nodes with pale blue or pinkish white flower. The chief chemical constituents present in the plant are aspartic acid, glycosides, saponins, stigmasterol, α-alanine, β-sitosterol etc. Cyperus rotundus (Nagaramotha), belonging to family Cyperaceae, is an important herb in the Ayurveda. It is a perennial herb with dark green thin stem, the leaves are long and sharp, flower stem has a triangular cross-section, the flower is 2 to 8 inch in length. The chief chemical constituents present in the plant are isohamnetin, khellin, p-coumaric acid etc, Volatile oil obtained from rhizome contains various terpenoids such as caryophyllene, camphene, copaene, cyperene, cyperenone, cyperol, cyperotundone, 180

3 cyperolone, D-copadiene, D-epoxyguaiene, isocyperol, isokobusone, kobusone, limonene, linoleic acid, linolenic acid, mustakone etc. Pharmacognostic Study All selected drugs for the present work, Bacopa monnieri, Emblica officinalis & Cyperus rotundus, were collected, cleaned and dried. The collected specimen were authenticated at National Botanical Research Institute (NBRI), Lucknow vide the specimen No NBRI-SOP-202 dated 29/07/2010 and the voucher specimen was preserved in the National Botanical Research Institute (NBRI) Lucknow for future reference. The mophological characters of the collected plant parts were examined for appearance, colour, odour and other specific characters, which were compared with the descriptions available in the literature and found the similarity. Further, the microscopical examination of the selected three drugs were performed in order to study the tissue arrangement in the cut sections. The transverse section of leaf and root of Bacopa monnieri, section of fruit of Emblica officinalis and transverse section of rhizome of Cyperus rotundus were examined to record their cellular/tissue arrangement and the findings were compared with the text descriptions. Further, the powder microscopic study of rhizome of Cyperus rotundus was carried out to ascertain the genuinity of the drug. These macroscopical and microscopical studies were carried out to assist in proper identification and authentication of the collected drugs. All three drugs were subjected to determination of various physical constants such as, loss on drying, ash values like total ash, water soluble and acid insoluble ash and extractive values like water soluble and alcohol soluble extractive values. These studies were carried out following the standard procedures as described in pharmacopoeia. The study results were recorded and the figures found to be within limit as per the Ayurvedic pharmacopoeia. Phtyochemical screening provide information pertaining to availability of phytoconstituents in a plant drug. Presence of various constituent imply effectiveness of the possible biological activities. Phytochemical screening of Cyperus rotundus rhizomes revealed the presence of aromatic acids, terpenoids, reducing sugars, alkaloids, phenolic 181

4 compounds, saponins, tannins, flavonoids, phytosterols and absence of xanthoproteins. Similarly, phytochemical screening of Bacopa monnieri plant found to contain some constituents such as: alkaloids, carbohydrates, flavonoids, glycosides, saponins, tannins and triterpinoids. Also the phytochemical screening of Emblica officinalis fruits showed the presence of alkaloids, carbohydrates, fixed oils, flavonoids, proteins, tannins and vitamin C. Formulation Study Extraction of plant parts The extraction of plant drug was carried out for obtaining the phytoconstituents available in the drug. For this purpose the fresh plant part was dried thoroughly after proper washing to remove earthy matters and other impurities. Efficient drying of a drug assist in proper extraction, which could be possible by cutting down into smaller pieces, i.e. the fleshy fruit of Emblica officinalis and rhizome of Cyperus rotundus required size reduction for rapid drying. The dried plant part was powdered and subjected to soxhlation process, in which drug was extracted continuously until removal of all phytoconstituents from the drug was achieved. The process was initiated by defatting the drug with petroleum ether (60-80 C) and the marc was again extracted using ethanol (95%) till completion of extraction. The solvents were recovered under vacuum from respective extracts, which were further dried on water bath at 60 C and were subjected to further studies. Extraction and characterization of volatile oil Freshly collected rhizomes of Cyperus rotundus, 1 kg, were washed properly, dried in shade and crushed to coarse powder before subjected to hydro distillation in a Clevenger apparatus for 4 hours (in accordance with B.P specification). To the drug 2.5 L of deionised water was added in a 5 L round bottom flask and heated to boiling, after which the volatile oil was evaporated along with water and finally collected in a collecting vessel after condensation. On condensation the volatile oil was separated as the upper phase which was collected and the lower phase was discarded. The isolated volatile oil was a colorless to light brownish yellow color liquid having camphoraceous odour. It was preserved in a sealed container and stored in cool dry place. The percentage yield of the volatile oil was approx. 0.2%. 182

5 Gas chromatography and Mass spectroscopic study of oil of Cyperus rotundus rhizome: The volatile oil extracted from Cyperus rotundus was analysed for the compounds present in it. The oil was characterized by the chromatographic techniques such as GC- MS and LC-MS. GC-MS was carried out at National Botanical Research Institute, Lucknow (U.P.) and LC-MS was carried out at Chemistry Department of Indian Institute of Technology, Kanpur (U.P.) using Water Q. TOF Premium HAB 213 Instrument. In GC analysis of the test fraction of volatile oil of Cyperus rotundus gave approx. 35 prominent peaks. The most abundant peaks with retention time of 8.25, 1182, 12.86, 13.94, 14.31, 15.58, 15.9, 16.22, 16.84, 18.64, 18.98, 19.54, 19.71, 19.89, 20.44, 20.56, 20.66, 21.77, 23.08, 23.91, 24.6, 25.06, 26.25, 27.15, 27.88, 28.2, 29.61, 29.83, 30.86, & were observed along with other peaks with smaller abundant. Out of the six more abundant peaks, the peak at and showed the response for cyperene and rotudene with maximum concentration of and 11.02% respectively. While the other peaks showed the response for cyperotundone, α-copaene, β-caryophyllene, epoxide, β- vetivone, γ-gurjunene & cyclopentene, 3-methyl-1-(OTMS). Screening of hair growth potential of drug extracts The powdered plant parts of Bacopa monnieri, Cyperus rotundus and Embilica officinalis were subjected to extraction and the extracts were employed to assess their individual hair growth potential. The ethanolic extracts of all these plants in varying concentrations of 1%,2%, 3% & 5% were taken and evaluated individually for their hair growth activities by applying the crude extracts on denuded skin of Wister albino rats and the results were recorded at the end of 30 days of treatment. On the basis of observations and the results shown by the individual extract for their hair growth activity it was found that 2%, 3% and 5% ethanolic extracts of Bacopa monnieri, Cyperus rotundus and Embilica officinalis showed promising hair growth promotion properties. These observations were taken into account during optimization of formulation of hair creams and hair oil of these three drug extracts. Simultaneously the volatile oil obtained from Cyperus rotundus rhizome was also evaluated for its hair growth activity at various concentrations of 1%, 2%,3% & 5% and it was observed that 5% volatile oil showed optimum result after 20 days of treatment. 183

6 Therefore, the 5% volatile oil alone was considered for the formulation of hair oil using oil bases. Development of formulations and characterization Based on the preliminary study of individual drug extracts it was observed that ethanolic extracts of all three drugs showed significant effect on hair growth promotion. Thus, its extracts were utilized for preparation of hair formulations in form of hair cream and hair oil. Formulation of hair creams Glyceryl monostearate, bees wax, mineral oil (light liquid paraffin), cetyl alcohol, glycerol, preservatives (methyl paraben and propyl paraben) are the various components used for preparation of cream base. The oil phases, glyceryl monostearate, cetyl alcohol, bees wax and propyl paraben were weighed individually according to the formula and taken in a clean porcelain dish. It was then kept over the heating mantle and allowed to melt. The calculated amount of herbal extracts were mixed with mineral oil and added to the molten oily phase with stirring. Then the mixture was heated upto 75 C. In another dish glycerol, methyl paraben and water were taken and heated to 75 C. At this temperature the oil phase was added slowly to the aqueous phase. Finally the dish was removed from the mantle and the molten base was stirred well using mechanical stirrer while cooling. At 45 C the formed cream was transferred to the container and stored. To find the most effective combination, the obtained three drug extracts were mixed in different proportions to prepare three formulations named as HCF1, HCF2 & HCF3. Characterization of hair cream formulation Stability studies: 1) Physical stability The prepared formulations were stored at room temperature and at elevated temperatures 25 ±2 C and 45 ±2 C for 180 days or 06 months. After two weeks of interval the formulations were tested for their physical characters like color, odour, visual appearance and consistency. In case of all three formulations HCF1, HCF2, HCF3, there was no considerable change in characters like color, odour, and consistency and there was no phase separation observed during the course of study at 25 o C. Whereas slight precipitation 184

7 was observed at 45 o C after 4 weeks and complete phase separation was observed after 6 months. 2) Chemical stability: Rheological studies (Spreadability) All three formulations HCF1, HCF 2 and HCF 3 were stored at room temperature and at elevated temperature 25 C and 45 C ± 2 C. At two weeks interval the samples were taken from each formulation and spreadability was determined using the following procedure. A glass plate was fixed to one end of the wooden scale and a small pulley was fixed to another end. A thread was fixed to a glass slide and passed over pulley. The slides were washed, dried and an excess of cream was placed between the slide and the glass plate. A 1Kg weight was allowed to rest on them for 5 minutes to expel air between them and provide for a uniform film of cream. Excess cream was scrapped off from the edges of the slide and the glass plate. The slides were subjected to pulled by 80gm weights. The time required to separate the slide from the glass plate completely (7.5 cm travel distance) was found out. Formulation of hair oil Different hair oil formulations at ratio of 1: 2 : 3, 2 : 1 : 3 & 3 : 2 : 1 of three drug extracts containing 5% ethanolic extract were prepared. Six hair oil formulations HO1, HO2, HO3, HO4, HO5 and HO6 were prepared out of which HO1, HO2 and HO3 were containing the 5% ethanolic extract of Bacopa monnieri, Cyperus rotundus and Embilica officinalis while HO4, HO5 & HO6 were containing 2%, 3% and 5% volatile oil obtained from Cyperus rotundus rhizome. All these formulations were prepared by pillow pouch method and direct boiling method using coconut oil as the base. Evaluation of hair oil preparation The prepared oils HO1, HO2, HO3, HO4, HO5 and HO6 were further studied for its physical stability parameters like specific gravity, acid value, saponification value, viscosity and ph. All these parameters were evaluated and found within the limit. 185

8 Biological Study of Formulations The biological study of various prepared formulations were conducted in accordance with the CPCSEA guidelines while handling experimental animals and the research protocols were approved by the institutional animal ethical committee (IAEC) which was approved by CPCSEA vide letter NO : 1122/ac/07/CPCSEA. Initially skin irritation test was carried out in order to evaluate the effect of the prepared formulation on the skin and to evaluate whether the creams (HCF 1-3) and oils (HO1-6) have any irritating effect on the skin. The test was carried out on Wister albino rats. None of the prepared formulation showed any erythema and/or edema, indicating that the prepared formulations were non-irritant to the rat skin. In order to evaluate the activity of formulated drugs, the desired pharmacological activity was assessed using suitable animal models. Hence, the prepared cream formulations HCF1, HCF2 and HCF3 and hair oils HO1, HO2, HO3, HO4, HO5 and HO6 were evaluated for the hair growth activity on Wister albino rats using the model developed by Uno The hair growth activity of the formulations were compared with a marketed formulation Coverit (2% minoxidil ethanolic solution), which was taken as a positive control. Both qualitative and quantitative studies were performed to evaluate the activity of the prepared formulation. In qualitative study, the time taken to restart the hair growth from the shaved area of skin and the time taken for maximization of hair growth was observed visually and recorded. Amongst all formulations of hair cream and hair oil, the HCF2, HO2 and HO6 showed promising hair growth activity, which was well comparable with the standard drug minoxidil. The hair growth initiation and maximization time of HCF2, HO2, HO6 and minoxidil found to be 4.66 & 16.33, 4.66 & 16.33, 5.33 & and 5.62 & days respectively. In quantitative analysis, the length of population of hair follicles in different cyclic phases were determined and the length of the each hair follicle was measured. On the 30th day of treatment, a maximized growth of hair was observed of which length was measured and recorded, which was quite noticeable. When compared with standard (minoxidil having hair length ±0.278) pertaining to hair growth activity, the formulations HO2, HO6 & 186

9 HCF2 showed the predominant hair growth activity in terms of hair length of ±0.115 mm, ±0.082 mm and ±0.071 mm respectively. These results clearly indicate that the developed formulations HO2, HO6 & HCF2 possess better hair growth promoting activity and are as effective as minoxidil (standard) and hold promising of a potential herbal alternative for minoxidil. It's anticipated that a number of fatty acids, e.g. palmitic, oleic, linoleic and linolenic and a combination of these acids exhibit a significant anti androgenic effect owing to their testosterone 5α reductase inhibitory activity (Liang and Liao, 1997; Yoon et.al., 2009) which are present in the selected medicinal plants. Flavonoids and triterpenoids also possess hair development promoting property by means of strengthening the capillary wall of the smaller blood vessels imparting hair follicles, improve blood circulation to nourish the hair follicles and there with the aid of promoting hair growth (Kobayashi et.al., 1993; Adhirajan et.al., 2003). Flavonoids have pleasant role by means of stimulating telogen to anagen phase, a method concerned in hair progress, and likewise rationale expressions of some development factors, similar to insulin like growth factor-1, vascular endothelial development factors, keratinocyte growth factors and hepatocyte development factors, all of which has stimulatory results on hair development (Tahahashi et.al., 1999; Seok - Seon et.al., 2002; Awe and Makinde, 2009). Minerals and selected amino acids may also have significant reasons for the hair growth activity but in addition devoid of potential side effect as compared to synthetic medications (Banerjee et.al., 2009). (Table 4.2, 4.3 & 4.5) Natural hair cream and hair oil formulations of Bacopa monnieri, Emblica officinalis & Cyperus rotundus on topical application reduced the time required for hair progress initiation and maximization period and was comparable to standard (2 % ethanolic Minoxidil solution) (Table 6.16). The quality of hair cream and oil formulated of Bacopa monnieri, Emblica officinalis & Cyperus rotundus in treated group was as good as the minoxidil treated group, resulted in production of gentle, soft and silky hairs. The utilization of synthetic medicines like Minoxidil and Finasteride as approved and permitted by FDA (Goodman and Gilman, 1996; Libecco and Bergfeld, 2004) have been abbreviated due to their high risk and side effect results. Treating of hair disease like alopecia for 5 years with minoxidil, it has been observed that the improvement has been shown to peak for one year with a slow decline in regrowth over subsequent years. These 187

10 crisis lead to the need and search of alternative natural product form herbs or plant origin possessing capabilities for hair growth and its development activities. The current research work has an outcome in accordance with many prior studies and findings for hair growth promoting activities of medicinal plants. 5% ethanolic extract formulations i.e. hair oil & hair cream, the formulations which have shown excellent results are HO2, HO6 & HCF2. They have resulted in significant increase in hair length and its thickness which are recorded in Table :6.17, 6.18, 6.19,6.20, 6.21 & 6.22 (Figure 6.10 to 6.54). Methanolic extract and its water insoluble fraction of green tea showed the presence of phenolic compounds. EGCG (epigallocatechin-3-gallate), a fundamental constituent of polyphenols of green tea was suggested to be useful in the therapy of androgenetic alopecia by selectively inhibiting 5α reductase activity (Kwon et.al., 2006). It would be viewed that the phenols present within the extract are liable for hair development promoting activities. Emblica officinalis extracts are a common ingredient present in most of the marketed hair oils, shampoos, conditioners and hair dyes formulations. When Bacopa monnieri, Emblica officinalis & Cyperus rotundus extracts were combined with coconut oil as the base and applied to the hair, the hair became dark, silky and lustrous. Therefore, it has been observed that Bacopa monnieri, Emblica officinalis & Cyperus rotundus hair oil also prevent in greying of hair. Further, Bacopa monnieri, Emblica officinalis & Cyperus rotundus extracts are a source of nutrition & vitamin for the scalp and due to this they prevent hair loss. When the hair oil containing these three drugs is massaged into the scalp, the rate of blood flow is increased and this increase in blood circulation causes the hair roots to turn out to be more stronger (Aclepsa.Com, 2007). Bacopa monnieri contains saponins due to which it had been used since thousand of years as hair a hair tonic and hair wash. Emblica officinalis contains flavonoids, vitamin C and tannins. Flavonoids have exceptional function and they stimulate telogen to anagen phase, an approach involved in hair growth and development. One of the drug Cyperus rotundus rhizome comprises essential or volatile oil, flavonoids and triterpinoids (Table 6.5) which attributes hair growth development activity through strengthening the capillary wall of the smaller blood vessels supplying blood to hair follicles, strengthen blood 188

11 circulation to nourish the hair follicles, thus, potentiate and improves the hair growth activity. Study of Krishnamoorthy et.al., (2010) clearly shows that the gel system containing extracts of Phyllanthus emblica, Bergamiia koenigii, Lawsonia alba, Indigofera tinctoria and Eclipta prostrate has hair growth promoting activity and it is as good as the synthetic formulation available in the market and it could be used as an alternative to minoxidil 2 % solution. Thus, due to the fact and legacy of usage of the three selected plant drugs for potentiating the hair growth activity and the preparation of poly herbal formulations, which are safe, effective and economic, will be able to manage the hair care problem in the society. Scanning Electron Microscopic (SEM) Study In the present study the scanning electron microscopic (SEM) analysis of the newly grown up hair was undertaken for determining the hair thickness in nanometre and to study its nano structure. It was also performed at IIT Kanpur using MIRA 3 TESCAN. The result obtained from the SEM analysis showed that there was a significant attainment in the hair thickness for the formulation HO2 ( to µm), HO6 ( µm) and HCF2 ( µm) as compared to the standard minoxidil 2% solution (124.91µm). 189

12 Conclusion The utilization of herbal cosmetics has improved many folds in personal hygiene and healthcare system. Hence, there is a tremendous demand for the herbal cosmeceutical individual care or personal health care industry, which is presently focusing and paying extra attention on the development of herbal-based cosmetics. As nowadays, it is a fast developing segment with a mammoth scope of manifold growth in coming years. Use of bioactive ingredients or phytoconstitutents in cosmetic formulations have beneficial effect on body features and provide nutrients, which are essential for maintaining healthy and beautiful skin or hair. At last it could be concluded that, the effect of Bacopa monnieri, Emblica officinalis & Cyperus rotundus polyherbal hair cream and hair oil formulations have shown the significant qualitative and quantitative hair growth activity both in length wise as well as in thickness wise. These effects are well comparable with the standard (minoxidil 2% solution ) and control group treated animals. The quantitative effect and influence of formulated polyherbal hair cream and hair oils containing extracts of Bacopa monnieri, Emblica officinalis & Cyperus rotundus certainly assist in promotion of hair growth and development by inducing hair follicles within the anagen phase. On the basis of comparison between the minoxidil 2% solution and the Bacopa monnieri, Emblica officinalis & Cyperus rotundus formulations pertaining to hair growth activity studies, it's observed that the formulation containing 5% ethanolic extract of three plant drugs showed better hair development activity than the standard minoxidil 2% solution. Even the hair oil formulation containing 5% volatile oil extracted from the Cyperus rotundus rhizome has also showen excellent hair growth activity. Future Scope: In future the prepared formulations can be utilized for the study on other animal models to further justify the hair growth and to treat some diseases related to hair like all types of alopecia (AA, AGA, AT, AU), dandruff, drug induced hair loss, male pattern hair loss (MPHL), female pattern hair loss (FPHL) etc. These preparations can also be used on human volunteer who are suffuring from hair disease like alopecia (AA, AGA), drug induced hair loss or chemotherapy induced alopecia (CIA). 190