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ORIGINAL ARTICLE
Year : 2013  |  Volume : 3  |  Issue : 4  |  Page : 367-374

Phytochemical, antimicrobial investigation and formulations development of extract of Semicarpus anacardium seed for their antidandruff potential


1 Department of Pharmacognosy, J. L. Chaturvedi College of Pharmacy, Nagpur, Maharashtra, India
2 Department of Pharmacology, Radharaman College of Pharmacy, Ratibad, Bhopal, Madhya Pradesh, India
3 S. L. T. Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Central University, Koni, Bilaspur, Chhattisgarh, India

Date of Submission09-Jun-2013
Date of Acceptance04-Jul-2013
Date of Web Publication15-Oct-2013

Correspondence Address:
Sandhya P Godi
Department of Pharmacognosy, J. L. Chaturvedi College of Pharmacy, Nagpur - 440 016, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.119848

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   Abstract 

Background: Dandruff is one of the most commercially exploited skin disease by the personal care industry all over the world, which affects 5% of the global population. Plant kingdom is a gold mine for novel and affordable skin care acting through novel mechanisms against skin pathogens. Identification of these remedies for such skin care is an important study for the preservation of traditional knowledge for the antimicrobial treatments. Semicarpus anacardium Linn. (Anacardiaceae) has been used for the skin care since ancient times. Aim of the Study: The aim of the present study was to investigate the antidandruff potential of S. anacardium seed extract and development of phytochemical, antimicrobial, and formulations. Materials and Methods: Antimicrobial activity of petroleum ether extract of S. anacardium seed was studied by agar well diffusion method using Escherichia coli, Staphylococcus aureus, and Candida albicans strains. Two herbal antidandruff shampoo formulations were developed and evaluated using parameters, physical evaluation, that is, color and consistency, pH, washability, homogeneity, spreadability, and skin irritation tests. The developed herbal shampoo formulations were assessed for their antidandruff potential manually on the human volunteers, respectively. One phenolic compound was isolated using preparative column chromatography, followed by thin layer chromatography. Results: Results of present studies revealed that there was a concentration-dependent antimicrobial effect compared with standard antibiotic disc, that is, gentamicin. S. aureus showed more zone of inhibition than E. coli. The inhibitory zone of all isolates was determined as 0-23 mm. The observed antimicrobial activity was due to the potent bioactive phytoconstituents present in the extract. Our formulations were found to be semisolid consistency, brownish black color with very slightly alkaline pH, that is, 7.3 and 7.1, which is compatible with normal skin physiology, easily washable, homogenous aggregations, good spreadabilty along with free from skin irritation. The developed antidandruff herbal shampoo formulations were manually checked for its safety and efficacy on human volunteers for a week and were found that complete irradication of dandruff within a time period of 7 days. These formulations were also softening the hairs. The preliminary phytochemical analysis of the petroleum ether extract of S. anacardium was showed the presence of phenolic compounds, tannins, saponins, flavonoids, steroids, glycosides, carbohydrates, and alkaloids. Further, two compounds were detected in the thin layer chromatographic analysis of extract of S. anacardium which having their R f values 0.9 and 0.6. One phenolic compound was isolated which has R f value 0.85. Conclusion: The petroleum ether extract of S. anacardium seed possessed a broad spectrum of activity against a panel of bacteria and fungi responsible for the most common microbial diseases. This plant can be used as a cheap source of active therapeutics. Further, exploration of plant derived antimicrobials as well as their identity is needed. However, further series of scientific studies are required to prove its clinical reliability, safety, and efficacy for the beneficial of mankind.

Keywords: Antimicrobial, formulations, phytochemical, Semicarpus anacardium seed


How to cite this article:
Godi SP, Mali PY, Rangari VD. Phytochemical, antimicrobial investigation and formulations development of extract of Semicarpus anacardium seed for their antidandruff potential. Int J Nutr Pharmacol Neurol Dis 2013;3:367-74

How to cite this URL:
Godi SP, Mali PY, Rangari VD. Phytochemical, antimicrobial investigation and formulations development of extract of Semicarpus anacardium seed for their antidandruff potential. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2018 Jul 20];3:367-74. Available from: http://www.ijnpnd.com/text.asp?2013/3/4/367/119848


   Introduction Top


Semicarpus anacardium Linn. (Anacardiaceae) popularly known as marking nut tree, which has many therapeutic applications in Indian system of medicine. [1] S. anacardium is medium size tree found in moist deciduous forests all over the country. [2] It has been used for medicinal and nonmedicinal purposes since ancient times. [3] Moreover, the plant is used by ayurvedic practitioners and traditional healers across the country albeit with caution. The stem, sap, fruit, and seeds are used by mankind for diverse purposes such as timber, paint, waterproofing, food, and medicine. [4] The oil is a powerful antiseptic and cholagogue. Ripe fruits are regarded as stimulant, digestive, nervine, and escharotics. Marking nut is a gastrointestinal irritant when taken by the mouth. Kernel is a good nutritive food, also appetizer, digestive, and carminative. It is a good cardiac tonic and a general respiratory stimulant. [5] S. anacardium have been reported to contain various phytoconstituents such as phenolic compounds like bhilavanol A (monoenepentadecyl catechol I), bhilavanol B (dienepentadecyl catechol II), anacardoside (glucoside), [6],[7] and important biflavanoids like semecarpuflavanone, jeediflavanone, galluflavanone, nallaflavanone, semecarpetic and anacarduflavanone, tetrahydromentoflavone, and so on have been isolated. [8],[9],[10] Furthermore, the preliminary phytochemical study showed the presence of alkaloids, saponins, tannins, flavonoids, steroids, glycosides, hexose sugars, diterpenes, mucilage, and gums were present in the extracts of leaves of this plant. [11] This plant also reported for its pharmacological potentials like hypolipidemic, [12],[13] hypoglycemic and antidiabetic, [14],[15] antiatherogenic, [16] antifungal, [17] antimicrobial, [18] antifertility, [19] neuroprotective, [20] cardioprotective, [21] anti-inflammatory, [9],[10],[22],[23] antiarthritic, [1],[24],[25],[26],[27] antioxidant, [28] immunomodulatory, [29],[30] antitumor, [31],[32],[33] and anticancer, [34],[35],[36] cytotoxicity, [37],[38],[39],[40] antimutagenicity, [41] brain toxicity, [42] and so on.

A number of multidrug-resistant strains and the appearance of strains with reduced susceptibility to antibiotics are continuously increasing due to the indiscriminate use of commercial antimicrobial treatment of infectious diseases. [43],[44] In addition, in developing countries, synthetic drugs are not only expensive and inadequate for the treatment of diseases but also often with adulterations and side effects. Therefore, there is a need to search new infection-fighting strategies to control microbial infections. [45] About 80% of individuals from the developed countries use traditional medicine, which has compound derived from medicinal plants. [46] Therefore, such plants should be investigated to understand their properties, safety, and efficacy and for a search of new potent antimicrobial compound and fractions. [47]

Dandruff is caused by skin irritation, diseases, or microbial infection. [48] It is a persistent and annoying common condition affecting the scalp. The etiology and various predisposing factors of the disease are still unknown; dandruff remains the challenge despite availability of therapeutic options. [49] Applications of herbal remedies, tattooing with herbomineral preparations are usual practices and have become part of several tribal rituals for skin problems including dermal inflammation, folliculitis (inflammation of follicles with infection by bacteria like Staphylococcus), and so on. Other common skin problems include, skin abuses, acne, perioral dermatitis, rosacea, subaceous cysts, and so on. [50] Multidrug-resistant bacteria including nosocomial pathogens have become important cause for higher skin care costs. A novel compound, with difference in mode of activity of antibiotics against microbes, is an attractive alternative against multidrug-resistant bacteria. [51] Nowadays natural medicine are gaining prominence, because they are economical, easily available, and relatively free from side effects. It is evident from the present scenario that herbal cure is gaining worldwide acceptance and has emphasized on modern scientific exploration, extraction, and evaluation of foil medicines from plants. These are either used directly as a plant extract or modified through further synthesis. [52] By keeping this view in mind, the present study was an effort to investigate phytochemical, antimicrobial, and formulations development of extract of S. anacardium seed for their antidandruff potential.


   Materials and Methods Top


Preparation of plant extract

Dried seeds of S. anacardium were purchased from the local market of Nagpur in the month of August 2007 and it was confirmed by Pharmacognocist, Dr. Vinod D. Rangari, Principal, J. L. Chaturvedi College of Pharmacy, Nagpur: 440 016, Maharashtra. The seeds were crushed into course powdered and extracted with petroleum ether (60-80°C) by maceration for a week. The crude oily semisolid residue mass were concentrated, stored, and preserved (2-8°C). The yield of extract was found to be 6.7% and it was used for phytochemical, antimicrobial, and formulations development for their antidandruff potential.

Procurement of bacterial species and dandruff

The standard bacterial strains such as Staphylococcus aureus,  Escherichia More Details coli, and Candida albicans used in the present study were procured from Rajiv Gandhi Biotechnology Centre, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur. Dandruff was collected from the scalp of human volunteers by swab technique using Sabourauds dextrose broth media. It was allowed to solidify and incubate the same for 48 h at 37°C. [53]

Thin layer chromatographic analysis of extract of S. anacardium

The preliminary phytochemical analysis of the petroleum ether extract was studied as per available procedures. [54],[55] The petroleum ether extract was subjected for thin layer chromatography (TLC) using conventional TLC glass plates, which was freshly coated with silica gel G, air dried, and activated it by keeping in an oven for 30 min at 110°C. The oily extract of S. anacardium was reconstituted in the methanol. 5 μL of this extract concentration was spotted on the prepared TLC plates (1.5-2 cm from one end of the plate) and developed in the mobile phase (Eluent), benzene:methanol (9.5:0.5). [56] Development of the chromatogram was done in a closed tank in which the atmosphere has been saturated with the eluent vapor. The plate was then dried under a stream of air to remove excess solvent. The visible bands were viewed under daylight after being sprayed with freshly prepared vanillin reagent. The plate was heated at 105°C for optimal color development. [57] The characterization of different compounds identified was done by calculating their R f values in the TLC system. [58] Briefly, the solvent front was marked on the TLC plates immediately after removing it from the chamber and allowed to dry before visualizing the bands relating to different compounds. The R f value was calculated by the following equation, R f value = distance moved by the component from the origin to spot centre/distance moved from origin to solvent front.

Preparative column chromatography of extract of S. anacardium

The oily petroleum ether extract of S. anacardium was chromatographed on a silica gel G column (65 ° 3 cm). Elution was performed using solvent mixtures of petroleum ether and benzene with increasing amount of benzene (100:0, 90:10, 80:20, 70:30, 60:40, 50:50). Successive fractions were collected and dried under vacuum using a rotary evaporator. All these fractions were subjected to thin layer chromatography using benzene (100%) as mobile phase. The fraction with petroleum ether:benzene (50:50) was produced single spot of a pure compound, which was isolated and subjected to Infra-red (IR) spectral analysis at Department of Pharmaceutical Sciences, R. T. M. University, Nagpur, Maharashtra for their identification, structural elucidation, and confirmation.

Antimicrobial activity of extract of S. anacardium

Antimicrobial activity of petroleum ether extract of S. anacardium seed was studied by agar well diffusion method, [59] using strains, S. aureus, E. coli, and C. albicans. Briefly, nutrient agar plates were swabbed with the test organism by following the procedure described in antibiotic susceptibility testing. Wells of diameter 4 mm were cut into the inoculated plates by strike cork borer. To the wells 10%, 20%, and 30% of the petroleum ether extract of S. anacardium seed were added and the plates were incubated at 37° for overnight. After incubation, the plates were analyzed for the zones of growth inhibition. The diameter of the zones of growth inhibition including the width of the wells was measured in millimeter (mm) and recorded. [60] The results were compared with the standard antibiotic disc, that is, gentamicin (10 μg/well) and expressed as mean ± standard error of the mean, each value replicate of three observations (n = 3).

Development of herbal antidandruff shampoo formulations

First herbal antidandruff shampoo formulation (Herbal antidandruff shampoo formulation-I) of petroleum ether extract of S. anacardium was prepared by dissolving the sodium hydroxide in a small quantity of water with heating at 75°C. Add biosulphur to the sodium hydroxide solution. Take sodium lauryl sulphate and stearic acid together and mix with heating at about 60°C and then add to the aqueous solution. Stir and cool, add perfume and preservative. The formula contains sodium lauryl sulphate paste (25 g), stearic acid (7 g), sodium hydroxide (1 g), biosulphur powder (2 g), water (65 mL), perfume [quantity sufficient (q.s.)], preservative (q.s.) and (quantitys sufficient) and 10%, 20%, and 30% strengths of petroleum ether extract of S. anacardium seed. The second shampoo formulation (Herbal antidandruff shampoo formulation-I) was formulated using the ingredients, selenium disulphite (2.5 g), bentonite (5 g), sodium lauryl sulphate paste (40 g), water (52.5 g), perfume (q.s.) and 10%, 20%, and 30% strengths of petroleum ether extract of S. anacardium seed. During preparation, first disperse the selenium disulphide evenly in bentonite. Mix sodium lauryl sulphate with water with heating and stirring to a temperature of about 90°C. Add part of this detergent mix to the selenium disulphide-bentonite dispersion with stirring to get a homogeneous mixture. Add this to rest of the detergent mix with continuous stirring. Cool to 40°C and add perfume. Addition of perfume is optional. [48]

Evaluation of developed antidandruff herbal shampoo formulations

Developed antidandruff herbal shampoo formulations of petroleum ether extract of S. anacardium was evaluated using below mentioned parameters.

Physical evaluation

Physical evaluation includes the checking of appearance, color as well as consistency by visual inspection. [61]

pH

pH of the formulations was measured by taking 1% aqueous solution of the formulations using digital pH meter. [62]

Washability

It was evaluated by applying formulations on the skin and then eases. The extent of washing with water was checked manually. [61]

Homogeneity

Formulations were tested for homogeneity by visual inspection, when after the formulation have been set into the container, spread it on the glass slide for the appearance, and tested for the presence of any lumps, flocculates, or aggregates. [61]

Spreadability

Spreadability was measured by using glass slides; the formulations were put into two slides by applying 1 kg weight on it for 5 min to compress the sample for uniform thickness. The time in seconds to separate slides was considered as measure of spreadability and it was calculated by using formula, [62] S = w l/t.

Where, S = spreadability in g-cm/s; w = weight of upper slide in g; l = length of slide in cm and t = time taken in seconds.

Skin irritation test

Skin irritation test was carried out on human volunteers. Three volunteers for one formulation were selected and applied 1 g of formulation on an area of two square inch to the back of the hand and observed for lesions or irritation. [61]

Assessment of antidandruff potential of developed herbal shampoo formulations

The developed herbal shampoo formulations of extract of petroleum ether of S. anacardium were assessed for their antidandruff potential manually on the human volunteers, respectively. The selected human volunteers were suffering from scaling of the scalp tissues. A total of 12 human volunteers were randomly divided into 03 groups of 04 volunteers in each, respectively. The developed herbal shampoos were applied gently by hand on the scalp. Keep it for 30 min and wash the scalp with normal tap water. Follow the same procedure to every day for a week and observe the scaling of scalp tissues.


   Results and Discussion Top


Though considerable advances are made in the pharmaceutical sciences, especially in synthetic chemistry, plants and their derivatives continue to maintain their significance in medicines. Increased interest in natural drugs than synthetic is because of a high degree of adverse side effects caused by the latter. [52] Therefore, our results are in-line with above statement, as preliminary phytochemical analysis of the petroleum ether extract of S. anacardium was showed the presence of phenolic compounds, tannins, saponins, flavonoids, steroids, glycosides, carbohydrates, and alkaloids. Further, two compounds were detected in the thin layer chromatographic analysis of extract of S. anacardium which having their R f values 0.9 and 0.6 by using benzene:methanol (9.5:0.5) as mobile phase as in [Figure 1]a. As we have isolated a compound which having R f value 0.85 using preparative column chromatography followed by thin layer chromatography in [Figure 1]b. The infra red spectrum (NaCl cell cm-1) of isolated compound along with interpretation as shown in [Figure 2] and was obtained in the wavelength region of 4000-400 cm- 1 . But, due to paucity of isolated compound, we have studied only infrared spectral analysis of the compound and it was confirmed as phenolic class of drugs of natural origin based on the interpretation of infra-red spectrum and comparison with the available literatures on the isolation of chemicals from the plant S. anacardium.
Figure 1: Thin layer chromatographic plates, (a) Petroleum ether extract of Semicarpus anacardium seed. (b) Isolated compound from the petroleum ether extract of Semicarpus anacardium seed

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Figure 2: FT-IR spectra of isolated compound from petroleum ether extract of Semicarpus anacardium seed

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Long before mankind discovered the existence of microbes, the idea that certain plants had healing potential, indeed, that they contained what we would currently characterize as antimicrobial principles, was well-accepted. Since antiquity, man has used plants to treat common infectious diseases and some of these traditional medicines are still included as part of the habitual treatment of various maladies. [63] Therefore, in agreement with above study, as we assessed antimicrobial activity of petroleum ether extract of S. anacardium seed by agar well diffusion method using E. coli, S. aureus, and C. albicans strains. The observed antimicrobial activity was due to the potent bioactive phytoconstituents present in the extract. In general, the plant extracts are much more active against gram-positive bacteria than gram negative. [64],[65] Results of our studies revealed that there was a concentration-dependent antimicrobial effect compared with standard antibiotic disc, that is, gentamicin as presented in [Figure 3]. This is due to phenolic toxicity to microorganisms which includes enzyme inhibition by the oxidized compounds possibly through reaction with sulfhydryl groups through more nonspecific interactions with the proteins. [66],[67] The density of lipopolysaccharide layer in the outer surface of bacterial cell wall is much lower in gram-positive bacteria when compared with that of gram negative. [68] So, certain antibacterial compounds can easily reach the peptidoglycan layer of the cell wall of gram-positive bacteria and penetrate into the cytoplasm. It causes the loose of the cells turgor pressure with a subsequent disorganization of the internal organelles. [69] Hence, the results of our study have in correlation with above finding, as S. aureus showed more zone of inhibition than E. coli. The inhibitory zone of all isolates was determined as 0-23 mm. The dimethyl sulfoxide (DMSO) control plate did not exhibit inhibition on the tested bacteria. The well diffusion method was preferred to be used in this study, since it was found to be better than the disc diffusion method. Therefore, the exhibited antimicrobial activity against gram positive, negative, and fungi supports folkloric use of this plant in the treatment of some skin diseases as broad-spectrum antimicrobial agents. The potential of developing antimicrobials from higher plants appears rewarding as it will lead to the development of a phytomedicine to act against microbes. Plant-based antimicrobials have enormous therapeutic potential as they can serve the purpose with lesser side effects that are often associated with synthetic antimicrobials. [70] With the current spread of antibiotic resistance almost at geometrical scale and obvious challenges confronted with by medical practitioners in the treatment of infectious diseases, proper attention should be given to such plants to reap the potential antimicrobial benefits inherent in them. [71],[72] Actual antimicrobial ingredients need to be extracted and identified, also its tolerable values in the human body as well as any toxic effects on human and animal tissues be investigated accordingly. [60]
Figure 3: Antimicrobial activity of petroleum ether extract of Semicarpus anacardium seed using agar well diffusion method

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Dandruff is one of the most commercially exploited skin disease by the personal care industry all over the world, which affects 5% of the global population. [73] Currently available treatment options for the management of dandruff include therapeutic use of zinc pyrithione, salicylic acid, imidazole derivatives, glycolic acid, steroids, and sulphur and coal tar derivatives. However, these agents show certain limitations, either due to poor clinical efficacy or due to the compliance issues. Furthermore, these drugs are unable to prevent recurrence. [74] Antidandruff shampoo is a very complex chemical system that contains primary, secondary, amphoteric and anionic surfacts besides conditioners, detangling agents, hair softeners, and antidandruff agents. Performance of an antidandruff agent in such a complex system is always an area of concern. The pH sensitivity, solubility, availability, and substantive deposition of the antidandruff agents on the scalp during shampoo wash are the key factors for determining the activity of antidandruff agents. Hence, the formulation of an antidandruff shampoo must fit into the above matrix of understanding to enable it to be effective against the causative organisms. [75] Varieties of antidandruff agents used widely in various antidandruff preparations such as climbazole, zinc pyrithione, octopirox, ketoconazole, selenium sulphite, coal tar, and so on. Among these, climbazole is one of the most popular antidandruff agents. It is an imidazole antifungal with well-proven safety data. [76]

Wide arrays of natural products from botanicals are traditionally in use over several hundred years. Plant kingdom is a gold mine for novel and affordable skin care acting through novel mechanisms against skin pathogens. [77] To overcome ailments or other problems of hair and also for decorative or beautification purposes, various hair care preparations are widely used. [48] Therefore, based on the antimicrobial effects of petroleum ether extract of S. anacardium seed, we have made a task to develop two antidandruff herbal shampoo formulations using the same extract. These formulations were evaluated for its safety and efficacy as per data presented in [Table 1]. Our formulations were found to be semi-solid consistency, brownish black color with very slightly alkaline pH, that is 7.3 and 7.1, which is compatible with normal skin physiology, easily washable, homogenous aggregations, good spreadabilty along with free from skin irritation. It is been revealed that the evaluation parameters lies within the limits as per the requirements for herbal antidandruff shampoos specified in the herbal standards. We have first time developed this type of herbal antidandruff shampoo formulations using the extract of S. anacardium seed which was manually checked for its safety and efficacy on human volunteers for a week and were found that complete irradication or clearance of dandruff within a time period of 7 days and about 100% of extract was effective for clearing the same. There is no recurrence of dandruff problem after the treatment with herbal formulations in the volunteers yet. Formulations also softened the hairs.
Table 1: Evaluation data of developed antidandruff herbal shampoo formulations of petroleum ether extract of Semicarpus anacardium seed

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   Conclusion Top


The global changing scenario is showing a tendency toward use of nontoxic plant products having good traditional medicinal background. The need or demand of such products has increased over the years. It shares a high portion of cosmetic products marketed. This plant can be used as a cheap source of active therapeutics. Further exploration of plant-derived antimicrobials as well as their identity is needed. The petroleum ether extract of S. anacardium seed possessed a broad spectrum of activity against a panel of bacteria and fungi responsible for the most common bacterial diseases. These studies will substantiate the traditional use of this plant in the various ailments and will be helpful for the ayurvedic formulation development in curing and treating diseases. However, further series of scientific studies are required to prove its clinical reliability, safety, and efficacy for the beneficial of mankind.


   Acknowledgment Top


Authors are thankful to the Principal and Management, J. L. Chaturvedi College of Pharmacy, Nagpur - 440 016, Maharashtra, India and Pinnacle Biomedical Research Institute, Bhopal for availing of the laboratory facilities during the course of investigation. Authors also wish to thank to Dr. Shekhar B. Waikar for his heartfelt encouragement and guidance during this study.

 
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