Journal of Pharmaceutical Negative Results

ORIGINAL ARTICLE
Year
: 2015  |  Volume : 6  |  Issue : 1  |  Page : 27--32

Insignificant activity of extracts of Gmelina asiatica and Ipomoea digitata against skin pathogens


Chandrika Mahendra1, D Vishakante Gowda2, U Venkata Babu1,  
1 Research and Development, The Himalaya Drug Company, Makali, Bangalore, India
2 Department of Pharmaceutics, JSS College of Pharmacy, Mysore, Karnataka, India

Correspondence Address:
Chandrika Mahendra
The Himalaya Drug Company, Makali, Tumkur Road, Bangalore - 562 162, Karnataka
India

Abstract

Objective: To study the antimicrobial activity of Gmelina asiatica aerial parts and Ipomoea digitata tubers against organisms causing acne, dandruff, and body malodor. Materials and Methods: The plant materials were extracted with petroleum ether, chloroform, ethyl acetate, and methanol using Soxhlet extraction unit. The extracts were tested for antimicrobial activity against Malassezia furfur, Propionibacterium acnes, and Corynebacterium diphtheriae at 1 mg/mL, 20 mg/mL, and 50 mg/mL concentrations, respectively. Results: The chloroform and ethyl acetate extracts of I. digitata showed mild antibacterial activity against C. diphtheriae at 20 mg/mL and 50 mg/mL concentration levels (13-14 mm), respectively and did not show any activity against P. acnes. The petroleum ether extract exhibited a zone of inhibition against C. diphtheriae at 50 mg/mL. The petroleum ether, chloroform, and ethyl acetate extracts of G. asiatica showed zones of inhibition in the range 12-13 mm, and of P. acnes and C. diphtheria, at 50 mg/mL. None of the extracts showed activity against M. furfur. The methanol extracts of both the plants failed to show any activity against all the selected organisms. Conclusion: The study showed that the plant extracts exhibit weak antimicrobial activity at the levels tested. Therefore, the extracts evaluated in this study cannot be used as alternatives to existing antimicrobial agents for acne, dandruff, and body malodor.



How to cite this article:
Mahendra C, Gowda D V, Babu U V. Insignificant activity of extracts of Gmelina asiatica and Ipomoea digitata against skin pathogens.J Pharm Negative Results 2015;6:27-32


How to cite this URL:
Mahendra C, Gowda D V, Babu U V. Insignificant activity of extracts of Gmelina asiatica and Ipomoea digitata against skin pathogens. J Pharm Negative Results [serial online] 2015 [cited 2019 Oct 17 ];6:27-32
Available from: http://www.pnrjournal.com/text.asp?2015/6/1/27/157383


Full Text

 INTRODUCTION



Skin is one of the most important physical barriers of the human body. It acts as both a barrier and a carrier for topical delivery systems. [1] The infections of the skin that are within the scope of topical formulations include acne vulgaris, seborrheic dermatitis, dandruff, [2] ringworm, and contact dermatitis among others. Body malodor, produced by the biological degradation of apocrine sweat secretions by coryneform bacteria, though not considered an infection may cause major social issues. [3] Topical medication is the preferred mode of administration, as the effect is localized on the site of action and is safe. Several chemical agents are available to treat these conditions. Ketoconazole, zinc pyrithione, climbazole, and selenium sulfide are the most common antifungal agents used to treat dandruff. [2] Topical antibiotics and benzoyl peroxide are commonly used to treat acne vulgaris. [4],[5] Triclosan is another antibiotic used in various topical products including deodorants. [2]

Acne, dandruff, body malodor, and other such conditions are not lifethreatening diseases. However, their presence may cause low selfesteem and other social problems. Therefore, agents that are safe and can combat these conditions are needed. Due to the toxicity of the synthetic agents, there exists a demand for natural antimicrobial agents that are safe and effective.

The purpose of the present study was to investigate the effects of Gmelina asiatica and Ipomoea digitata on scalp and skin infections based on their folklore and traditional usage in the Indian system of medicine for these conditions. [6] The aim was to evaluate the extracts of the aerial parts of G. asiatica and the tubers of I. digitata using various organic solvents and to establish their efficacy in killing or inhibiting the growth of representative microorganisms such as Propionibacterium acnes, Corynebacterium diphtheriae, and Malassezia furfur.

 MATERIALS AND METHODS



Materials

Plant material, Soxhlet apparatus, petroleum ether, chloroform, ethyl acetate, methanol, Sabouraud dextrose agar, Sabouraud dextrose broth, sterile oil, peptone water, and antibiotic ketoconazole were procured from Himedia Laboratories, Mumbai, Maharashtra, India. Dimethyl sulfoxide (DMSO) was procured from E. Merck Ltd., Mumbai, Maharashtra, India.

Pharmacognostical characteristics of I. digitata tuber

The genus Ipomoea belongs to the family Convolvulaceae. I. digitata is an extensive perennial climber with large, ovoid, and tuberous roots. The leaves are large, palmate, five-to-seven-lobed, oval, lanceolate, acute or acuminate, glabrous, and with prominent nerves beneath. The flowers are widely campanulate, appearing in axillary corymbose cymes. [7],[8],[9]

Organoleptical characteristics

The nature of the I. digitata sample is tuberous; its odor: A characteristic foul smell; taste: Not characteristic; color: Dark brown outer skin with dull white inner part; and texture: Rough.

Macroscopic characteristics

The I. digitata sample used in the study contains pieces of tuberous roots, which are mostly square- or rectangular-shaped, measuring 1-4 cm. The pieces may be either shrunken at the center or completely shrunken with protruding or curved edges. The circles of vascular strands are clearly visible [Figure 1].{Figure 1}

Microscopic characteristics

The transverse section of the I. digitata tuber reveals an outer cork layer and an inner ground tissue. The cork layer is made up of three to four layers of rectangular-shaped cork cells. The ground tissue is made up of thin-walled, polygonal or irregular parenchyma cells. The region with visible vascular strands shows a few outer layers of cambial cells. The ray is broad and made up of undifferentiated parenchyma cells. The pith region is parenchymatous. A single vascular bundle shows numerous vessel elements, fibers, parenchyma cells, and a few crushed phloem cells on the top. In the cortical region, numerous rosette-form calcium oxalates are observed. Starch grains are single with central hilum.

Pharmacognostical characteristics of aerial parts of G. asiatica

The genus Gmelina belongs to the family Lamiaceae. G. asiatica is a shrub that grows to a height of 1-3 m, attaining a maximum height of up to 10 m, usually with spines and minute yellowish brown hairs on young shoots. The petioles are 0.5-4.5 cm long. The leaves are papery, ovate to obovate, 3-5 cm long and 2.2-3 cm wide (growing up to 9 cm long and 8.5 cm wide), abaxially dark brown, villous and glandular, adaxially glabrescent, base cuneate, margin entire or lobed, apex acuminate, veins present in three to four pairs. Inflorescences are terminal, usually pendulous, with a few to many flowers. Primary floral branches are very short, with one to five flowers. The flowers are pendulous, with calyx measuring 3-6 mm long and 2.5-4 mm wide, densely dark brown, pubescent, with two to several disc-shaped glands outside, and glabrous inside. The corolla is yellow, 2-5 cm, two-lipped and four-lobed, lower lip three-lobed, upper lip entire, with scattered appressed hairs outside and dense glandular hairs inside. Filaments have dense glandular hairs. Ovary is four-locular and glabrous. The style is slender, hardly exerted, and apically curved. The stigma has two unequal clefts. Drupes are yellow, ovoid to obovoid, and glabrous. Bracts are leaflike, small to large. [10]

Organoleptical characteristics

The nature of the G. asiatica sample is: Aerial parts; its taste: Not characteristic; odor: Not characteristic; and color: Grayish brown to brown.

Macroscopic characteristics

A market sample of G. asiatica consists of dried aerial parts, including stem pieces. Fragments of leaves may also be present in some batches. However, the present report is on stem pieces only. Stem pieces are up to 8 cm long and 2 cm thick. They are dark brown to grayish black in color. Numerous dots of pale gray lenticels are present on the surface. Many of the matured pieces have grayish longitudinal streaks on the surface. Cut ends are yellowish to mild brown or reddish brown in color. Hollow pith is observed in almost all the thicker stem pieces. Very young stem pieces have spines up to 5 mm long in the nodes. On close observation, cut ends show longitudinal rays and circles of annual rings [Figure 2]. [11]{Figure 2}

Microscopic characteristics

The transverse section of a matured stem shows a typical dicot structure with outer fissured or cracked cork made of suberized cells with lenticels followed by the secondary cortex, secondary phloem, wood, and central pith. The secondary cortex is parenchymatous with starch grains as cell inclusions. The secondary phloem consists of patches of bundle cap fibers from primary vascular bundles, alternatively with groups of a few macrosclereids, which have tiny lumen and other phloem elements. The wood consists of vessel elements, fibers, and tracheids. Annual rings are clearly visible, demarcated with thin tracheids and narrow lumen. The rays are mostly present in two layers. Pith cells below the xylem elements are larger, with a hollow inner part. Parenchyma cells of the secondary phloem, ray cells, and pith cells are filled with starch grains and calcium oxalates. Starch grains are simple or compound, without clear hilum, and up to 12.5 μm in size. Calcium oxalates are prismatic, square, or rectangular, and are abundant in the pith cells and ray cells. The radial longitudinal section and the tangential longitudinal section of the stem also reveal the characteristic dicot structure.

Preparation of the plant extract

Soxhlet apparatus was used to conduct successive extraction. The dried plant material was used for extraction. Initially, the required quantity of the material was packed into the thimble and 500 mL of solvent used for extraction was poured into the flask. The cycle was repeated continuously (it can be sustained as long as needed). The Soxhlet extraction was performed for 18-24 h, until the siphon tube collected clear solvent. Later, the extracted solvent was evaporated under reduced pressure to get the dried extract.

Phytochemical analysis

Chemical tests for the screening and identification of bioactive chemical constituents in I. digitata and G. asiatica were carried out with the extracts using the standard procedure as described. [12],[13],[14]

Antimicrobial screening

Microorganisms

The microbial cultures of M. furfur, P. acnes, and C. diphtheriae were procured from the National Centre for Industrial Microorganisms (NCIM), Pune, India.

Subculture and standardization

Preparation and standardization of stock cultures of M. furfur

Cultures on receipt were subcultured on sterile oil-coated Sabouraud dextrose agar plates and further stored in slants as stock cultures. For the experiment, stock culture was prepared by inoculating each culture from slants to flask in sterile Sabouraud dextrose broth, and a drop of oil was added and incubated at 30°C for 48 h in microaerophilic conditions. The stock culture was adjusted to 0.11 OD by spectrophotometer at 650 nm and used for assay.

Preparation and standardization of stock cultures of P. acnes

Cultures on receipt were subcultured on sterile reinforced clostridial broth and further subcultured in slants prepared with the same media. The cultured slants were stored at 4°C for further use. For the experiment, stock culture was prepared by inoculating culture from slants to flask in reinforced clostridial broth at 37°C for 48 h in anaerobic conditions. The stock culture was adjusted to 0.5 McFarland standard and used for assay.

Preparation and standardization of stock cultures of C. diphtheriae

C. diphtheriae was subcultured on trypticase soy agar plates and further subcultured in slants prepared with the same media. The cultured slants were stored at 4°C for further use. For the experiment, stock culture was prepared by inoculating each culture from slants to flask in sterile trypticase soy broth incubated at 37°C for 24 h. The stock culture was adjusted to 0.5 McFarland standard and used for assay.

Preparation of resazurin solution

The resazurin solution was prepared by dissolving 270 mg of the dye in 40 mL sterile distilled water. A vortex mixer was used to prepare a homogenous solution.

Determination of zone of inhibition

Sterile agar plates were prepared with their respective media and 0.1 mL inoculum from standardized culture of test organism was spread uniformly. Wells were prepared using a sterile borer of diameter 10 mm to obtain the desired test concentration; 100 μL of the test substance and a standard antibiotic (ciprofloxacin for bacteria and ketoconazole for fungi) were added in each well separately. The plates were placed at 4°C for 1 h to allow diffusion of the test solution into the medium. Then, they were incubated at 37-28°C for a period of time sufficient for the growth of at least 10-15 generations, that is, 24 h for bacteria and 48 h for fungi. The zone of inhibition of microbial growth around the well was measured in mm. [15],[16],[17],[18]

 RESULTS



Phytochemical analysis

The yields of extracts were recorded as mentioned in [Table 1]. [Table 2] and [Table 3] show the summarized phytochemical screening of chemical constituents present in the extracts of I. digitata and G. asiatica under study on qualitative basis. The results revealed the presence of active compounds in different extracts. The extracts of I. digitata exhibit the presence of carbohydrates, glycosides, flavonoids, phenolics, tannins, phytosterols, and triterpenoids. However, saponins, alkaloids, fixed oil, and fats were absent in the extracts of I. digitata [Table 2]. The extracts of G. asiatica exhibit the presence of carbohydrates, alkaloids, glycosides, flavonoids, phenolics, tannins, phytosterols, and triterpenoids. However, saponins, fixed oil, and fats were absent in the extracts of G. asiatica [Table 3].{Table 1}{Table 2}{Table 3}{Table 4}

The analysis of plant extracts revealed the presence of phytochemicals, which are known to exhibit medicinal and physiological activities.

Antibacterial activity

Results of the study indicated that the extracts exhibited maximum activity against C. diphtheriae. The chloroform and ethyl acetate extracts of I. digitata showed mild antibacterial activity against C. diphtheriae at 20 mg/mL and 50 mg/mL concentration levels and did not show any activity against P. acnes and M. furfur. The zone of inhibition for C. diphtheriae at 50 mg/mL ranged 13-14 mm. The petroleum ether extract showed a mild activity against C. diphtheriae at 50 mg/mL, whereas the methanol extract failed to show any activity at the levels tested [Table 4].

The petroleum ether, chloroform, and ethyl acetate extracts of G. asiatica showed mild activity against P. acnes at 50 mg/mL, the zone of inhibition varying 12-13 mm [Table 4]. The same extracts showed slightly better activity against C. diphtheriae at 20 mg/mL and 50 mg/mL, with the zone of inhibition varying 11-13 mm. The methanol extract failed to show any activity at the levels tested. The yield of extracts [Table 1] showed that the methanol soluble principles were at higher levels, 6.3% for I. digitata and 9.93% for G. asiatica. However, antibacterial activity was found to be weak in both the plants. The principles responsible for the weak activity shown could be due to nonpolar components of the plant.

 DISCUSSION



The present work demonstrated the weak antimicrobial activity of G. asiatica aerial parts and I. digitata tubers against the selected skin pathogens P. acnes and C. diphtheriae. The results of the study also indicated that the two extracts' efficacy against M. furfur is negligible. The observed inhibition of P. acnes and C. diphtheriae suggests that the nonpolar components of the plant could probably suppress the bacterial skin pathogens if purified further. The folkloric use of I. digitata and G. asiatica for skin and scalp infections could have been due to this activity. However, this would require further research to correlate and identify the principle with the activity, and a combination of principles acting synergistically should be evaluated. The probability of obtaining a fraction for dandruff inhibition does not seem to be high. Therefore, the extracts evaluated in this study cannot be used as alternatives to existing antimicrobial agents for acne, dandruff, and body malodor.

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