HAIR GROWTH STIMULATING EFFECT AND PHYTOCHEMICAL EVALUATION OF HYDRO-ALCOHOLIC EXTRACT OF GLYCYRRHIZA GLABRA

Abstract

In this particular study, Hydro-alcoholic extract of Liquorice was evaluated for its use in Alopecia, where the extracts were compared with the activity of Marketed drug Minoxidil and the tests were carried out on Female Albino Rat. The Results were very much encouraging as 2% Hydroalcoholic extract has shown a better hair growth than that of the marketed drug Minoxidil. The extract was also subjected to preliminary Phytochemical Evaluations whereby it has shown the presence of Coumarines, Saponins, Phytosterols and Flavonoids along with Carbohydrates, Starch and Fixed Oils. TLC of the extract using Pre Coated Silica gel GF Plate while detected under UV have shown 1 Spot (Rf:0.67) with Ethyl acetate as Mobile Phase, 1 Spot (Rf:0.44) with Benzene: Toluene (4:6) as Mobile Phase and 1 Spot (Rf:0.96) with Benzene: Chloroform (3:7) as Mobile Phase.

Headnote

ABSTRACT

In this particular Study Hydro-alcoholic extract of Liquorice was evaluated for its use in Alopecia, where the extracts were compared with the activity of Marketed drug Minoxidil and the tests were carried out on Female Albino Rat. The Results were very much encouraging as 2% Hydro-alcoholic extract has shown a better hair growth than that of the marketed drug Minoxidil. The Extract was also subjected to preliminary Phytochemical Evaluations whereby it has shown the presence of Coumarines, Saponins, Phytosterols and Flavonoids along with Carbohydrates, Starch and Fixed Oils. TLC of the extract using Pre Coated Silica gel GF Plate while detected under UV have shown 1 Spot (Rf:0.67) with Ethyl acetate as Mobile Phase, 1 Spot (Rf:0.44) with Benzene: Toluene (4:6) as Mobile Phase and 1 Spot (Rf:0.96) with Benzene: Chloroform (3:7) as Mobile Phase.

KEY WORDS: Hair Growth, Glycyrrhiza glabra, Liquorice, TLC, Hydro-alcoholic extract.

INTRODUCTION

Hair follicle growth occurs in cycles. Each cycle consists of a long growing phase (anagen), a short transitional phase (catagen) and a short resting phase (telogen). At the end of the resting phase, the hair falls out (exogen) and a new hair starts growing in the follicle beginning the cycle again. Normally, about 40 (0-78 in men) hairs reach the end of their resting phase each day and fall out. When more than 100 hairs fallout per day, clinical hair loss (telogen effluvium) may occur. A disruption of the growing phase causes abnormal loss of anagen hairs (anagen effluvium) (Rudnicka L et al., 2008; Zhou Z.Y et al., 2007).

Alopecia means loss of hair from the head or body. Alopecia can mean baldness, a term generally reserved for pattern alopecia. Compulsive pulling of hair can also induce hair loss. Hairstyling routines such as tight ponytails or braids may cause traction alopecia. Both hair relaxer solutions, and hot hair irons can also induce hair loss. In some cases, alopecia is due to underlying medical conditions, such as iron deficiency (Rudnicka L et al., 2008). Generally, hair loss in patches signifies Alopecia areata. Alopecia areata typically presents with sudden hair loss causing patches to appear on the scalp or other areas of the body. If left untreated, or if the disease does not respond to treatment, complete baldness can result in the affected area, which is referred to as Alopecia totalis. When the entire body suffers from complete hair loss, it is referred to as Alopecia universalis. It is similar to the effects that occur with chemotherapy (Zhou Z.Y et al., 2007).

The nature remains as the potential source of organic structures of unparalleled diversity. The therapeutic use of Medicinal Plants has gained considerable momentum in the world during the past decade. The overuse of synthetic drugs with impurities, resulting in higher incidence of adverse drug reactions in more advanced communities, has motivated mankind to go back to nature for safer remedies. The selected plant Glycyrrhiza glabra was reported to have wide ethnomedical use (Shibata S. et al., 2000).

Minerva med reported that metabolic and toxic effects caused by prolonged daily ingestion of Liquorice are well known. Such acquisition doesn't seem to be known enough by practitioners and by common people. Besides it contains active substances such as Glycyrrhizin, steroids similar to the adrenocortical ones; among these the most important is Beta-Glycyrrhetinic acid. This, in vivo and in vitro, produces salt and water retention by means of a mineral-corticoid mechanism, and clear suppression of the Renin- Angiotensin-Aldosterone axis. A low plasmatic level of Renin and Aldosterone is a common feature. The clinical picture in many ways is similar to the primary Aldosteronism and for this reason the above mentioned syndrome is usually called "Pseudoaldosteronism" (Colloredo G et al., 1987). Saeedi .M et al. reported that Creams containing whole licorice (often combined with extract of chamomile) are in wide use as "natural hydrocortisone creams." However, there is only preliminary supporting evidence for this use. In one double-blind, placebo-controlled trial of 30 people, licorice gel at 2% was more effective than placebo or 1% gel for reducing symptoms of eczema (Saeedi M et al., 2003). Besides there were some ethno-medicinal claims regarding the use of Liquorice in Alopecia, which encouraged us to go for the study, and we have tried here to establish the ethno- medicinal claim regarding its use in Alopecia.

METHODS:

Place of Collection: The roots were collected from an established Crude Drug dealer from Kolkata and have been identified by Deptt. of Botany, Guwahati University vide Specimen No: 11746 and a Sample voucher has been deposited for further reference.

pH Determination:

a) pH of 1% Solution :

1 gm. of the accurately weighed drug was treated with 100 ml. of distilled water and filtered. pH of the filtrate was checked with a pH meter having standardized glass electrode.

b) pH 10% Solution :

10 gm. of the accurately weighed drug was treated with 100 ml. of water and filtered. pH of the filtrate was checked with a pH meter having standardized glass electrode.

Loss on drying:

A glass stoppered shallow weighing bottle was dried and weighed and 3 gm of the powdered drug was transferred to the bottle. The bottle was then stoppered and the bottle along with the contents was weighed. The sample was then distributed as evenly as practicable by gentle side wise shaking to a depth not exceeding 10 mm. The loaded bottle was then placed in the hot air oven; the stopper was removed and left it also in the oven. The powdered drug was then dried to constant weight or for 30 mm and at a temperature of 105°C. After drying was completed the hot air oven was opened and the bottle was closed promptly and allowed to cool at room temperature. The bottle and the contents were then weighed. The procedure was continued until a constant weight was obtained.

Ash value: (Pharmacopoeia of India)

a) Total Ash :

2 gm of air dried drug was weighed accurately in a tared silica crucible and incinerated at a temperature not exceeding 450°C until free from carbon, cooled and weighed. The percentage of ash with reference to the air dried drug was calculated.

b) Water Soluble Ash :

The ash was boiled for 5 minutes with 25 ml of distilled water and the insoluble matter was collected on an ash less filter paper, washed with hot water, and incinerated for 15 minutes at a temperature not exceeding 450°C. The weight of the insoluble matter was subtracted from the weight of the ash, the difference in weight represents the water soluble ash. The percentage of water soluble ash with reference to the air dried drug was then calculated.

c) Acid Insoluble Ash :

The ash was boiled for 5 minutes with 25 ml of 2M hydrochloric acid and the insoluble matter was collected in a Silica crucible or on an ash less filter paper, washed with hot water, incinerated, cooled in a desiccator and weighed. The percentage of acid insoluble ash with reference to the air dried drug was then calculated.

Extractive value:

a) Ethanol Soluble Extractive :

5 gm of the air dried drug was coarsely powdered, taken in a stoppered conical flask and macerated with 50 ml of ethanol (90%) for 24 hrs shaking frequently during the first 6 hrs and allowing standing for 18 hrs. Thereafter, it was filtered rapidly taking precautions against loss of ethanol, and then the filtrate was evaporated to dryness in a tared flat bottom shallow dish, dried at 105°C and weighed. The percentage of ethanol - soluble extractive was calculated with reference to the air dried drug.

b) Chloroform Soluble Extractive :

5 gm of air dried drug was coarsely powdered, taken in stoppered conical flask and macerated with 25 ml of chloroform for 24 hrs shaking frequently during the first 6 hrs and allowed standing for 18 hrs. Thereafter, it was filtered rapidly taking precaution against loss of petroleum ether, and then the filtrate was evaporated to dryness in a tared flat bottomed shallow dish, divides at 105°C and weighed. The percentage of petroleum ether soluble extractive was calculated with reference to the air dried drug.

C) Water Soluble Extractive:

5 gm of the air dried drug was coarsely powdered, taken in a stoppered conical flask and macerated with 50 ml of distilled water for 24 hrs, shaking frequently during the first 6hrs and allowing standing for 18 hrs. Thereafter it was filtered rapidly taking precautions against loss of chloroform water, and then the filtrate was evaporated to dryness in a tared flat bottom shallow dish, dried at 105°C and weighed. The percentage of water soluble extractive was calculated with reference to the air dried drug.

Drying and pulverization:

The collected plant material (Roots) was shade dried and then they were pulverized to coarse powder and passed through mesh size 40.

Preparation of extract by continuous hot extraction:

The roots were dried in shade and powdered to get a coarse powder. About 75 gm of dry coarse powder was extracted with water- ethanol (1:1) by continuous hot percolation using soxhlet apparatus (40-60°c). The extraction was continued for 7 days. The hydro-alcoholic extract then filtered and concentrated by vacuum distillation. A brown colour shiny residue was obtained.

Qualitative chemical evaluation:

Extract was subjected to various qualitative chemical tests for detecting the presence of various Phytoconstituents (Table No. 6). The procedures were followed as per the procedures laid down by Kokate C.K et al., 2008 and Khandelwal K.R, 2012.

Thin layer chromatographic separation:

For TLC, Precoated Silica Gel GF plates were used, by trial and error method various solvent systems were selected and spot visualization was done in UV chamber (Mukharjee P.K, 2010).

Hair growth stimulating activity:

Approval of the study:

The research protocol of the animal experimentation was approved by the "Institutional Animal Ethical Committee' of Girijananda Chowdhury Institute Of Pharmaceutical Science, Azara, Guwahati-17, Assam. GIPS/IAEC No. : GIPS BPH/2013/6

Collection of animals:

Animals (Wister Albino Rats) weighing 120- 150 g and aged 3-4 months were collected from Animal House of GIPS, Azara and used for Hair growth stimulatory study. The animals were handled according to CPCSEA Guidelines of Good Laboratory Practice. Animals were kept overnight in laboratory conditions acclimatize with the Laboratory environment.

Preparation of test sample:

The test sample was prepared by preparing suspension of the dried hydro-alcoholic extract. 0.5gm & 1gm extract were dissolved in 50ml ethanol (90%) each, which gave concentration of 1% & 2% solution respectively.

Hair growth stimulatory study:

A 4 cm2 area of the dorsal skin of the rats were shaved off using a marketed hair removal cream. The extract solution and Minoxidil (0.4 ml) was applied to the denuded area of the rat once a day. This treatment was continued for 10 days during and after which hair growth pattern was observed visually and recorded (Adhirajan N et al., 2003; Dattaa K et al., 2009).

RESULTS AND DISCUSSION:

Pharmacognostic Evaluations: (Table I)

pH of 1% solution was found to be 6 and that of 10% solution was found to be 5, which suggests the drug to be an acidic. Moisture Content was found to be 8%w/w. Total Ash, Acid insoluble ash and Water Soluble ash were found to be 7.9%, 2.07% and 5.67% respectively, when calculated with reference to the air dried drug. Water Soluble Extractive value, Ethanol Soluble extractive value and Chloroform Soluble extractive values were found to be 12.2%, 4.04% and 1.28% respectively, when calculated with reference to the air dried drug. These parameters help, in identification of the pure crude drug, while checking for adulterants. The results are shown in Table I.

Phytochemical Evaluation: (Table II)

The extract when screened for various Phytoconstituents and has shown the presence of, Coumarines, Saponins, Phytosterols and Flavonoids along with Carbohydrates, Starch and Fixed Oils, and are shown in Table II.

Thin layer Chromatography: (Table III)

When the extract was subjected to Thin Layer Chromatography, using Precoated Silica Gel GF plates as Stationary Phase and Choosing various mobile phases based upon the various Phytoconstituents found through phytochemical screening on a trial and error method, the extract has shown Single spots with, Chloroform, Benzene, Ethyl Acetate, Benzene :Toluene (4 :6) and Benzene : Chloroform (3 : 7) as Mobile Phases, while UV Detector was used for detection. The Results were recorded and Shown in Table III.

Hair growth stimulatory activity: (TABLE IV) (Fig.I)

After carrying out the study it was found that the hydro-alcoholic extract of liquorice showed a profound hair growth activity. Further it was also found that 2% concentration of the extract as compared with the standard drug used (Minoxidil 2%), has shown better hair growth activity. The Findings are shown in Table IV. and a graphical representation is also provided. Fig.I below which shows the Hair Growth observed after 10 days.

CONCLUSION:

The results indicate that Liquorices (Glycyrrhiza glabra) has a potent hair growth activity and after careful checking of other safety parameters it can be used in herbal formulations to treat various types of Alopecia.

ACKNOWLEDGEMENT:

The authors are very much grateful to the Management of Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati, Assam, India, for providing the facilities needed to carry out this Study.

Sidebar

Cite this article: Deb Roy Saumendu, Karmakar Prithivi Raj, Dash Suvakanta, Chakraborty Jashabir, Das Biswajit (2014), HAIR GROWTH STIMULATING EFFECT AND PHYTOCHEMICAL EVALUATION OF HYDRO-ALCOHOLIC EXTRACT OF GLYCYRRHIZA GLABRA, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 40-47

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Source of Support: NIL

Conflict of Interest: None Declared