Received date: November 25, 2016; Accepted date: December 17, 2016; Published date: December 20, 2016
Citation: Mehmood Z, Dixit AK, Singh A, Farooq S, Chhimwal SL (2016) Indian Herb Hemidesmus indicus - A Potential Source of New Antimicrobial Compounds. Med Chem (Los Angeles) 6: 734-738. doi: 10.4172/2161-0444.1000422
Copyright: © 2016 Mehmood Z, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The results of present study support the view that Hemidesmus indicus and its isolated bioactive compound (2H4MB) showing potential antibacterial and antifungal activities that could be utilized to control infectious diseases caused by the test pathogens and Candida albicans in the human system. Analysis of essential oil from root of the plant obtained by using Clevenger apparatus by hydro distillation was undertaken through GC-MS. 2-Hydroxy- 4-methoxybenzaldehyde (2H4MB) was the major chemical entity (99.41%) amongst 10 identified compounds. The in vitro antibacterial activity of 3 different extracts (Hexane, Methanol and Aqueous) and Bioactive compound 2-Hydroxy-4-methoxybenzaldehyde (2H4MB) as well as MIC values of isolated compound (2H4MB) were performed. The most active extract was found to be the hexane extract showing the maximum zone of inhibition of 22 mm against Staphylococcus aureus. The isolated bioactive compound (2H4MB) showed the highest diameter of zone of inhibition of 23 mm against Staphylococcus aureus. The Minimum Inhibitory Concentration values of the Bioactive compound 2H4MB on test pathogens varies from 80 μg/ml to 250 μg/ml, Minimum Bactericidal Concentration values varies from 150 μg/ml to 250 μg/ml. The MFC values and MIC values of 2H-4-MB on Candida albicans were 200 μg/ml and 150 μg/ml respectively. The active extract was also tested for cytotoxicity using freshly obtained sheep erythrocytes and it was revealed zero cytotoxicity.
Hemidesmus indicus; Antibacterial activity; Anticandidal activity; Bioactive compound
Almost 50,000 people killed every day due Infectious diseases which are the prominent cause of premature deaths. The most common pathogens for infections are Escherichia coli, Salmonella spp., Staphylococcus aureus. Drug resistance to common pathogens is the world major problem [1-3]. The microorganisms are now resistant due to continuous and indiscriminate use of antibiotics. Adverse reactions hypersensitivity, immunosuppressant and allergic reactions are also reported from antibiotics [4,5] which may associate with many clinical problems to treat infections . Now this is a high time to discover and develop new antimicrobial drugs to control infectious diseases caused by drug resistant microbes. One of the way is to evaluate and study the local herbs for their antimicrobial potentials. Medicinal plants always remain an ultimate source to treat serious diseases. As per WHO  estimates 80% of the population in the world is depend on the traditional therapies using plant extracts or their phytochemical constituents. Searching of new chemical entities from plants with antimicrobial properties taking advantage of recent biological and medical technologies is a new and developing field. Since a long time, medicinal herbs and their bioactive constituents like essential oils have been known for their antimicrobial properties [8-11]. In recent times, the search for potent antimicrobial agents has been credited to medicinal plants. A number of plants are useful as a medicine in the treatment of diseases in the body and in most of cases the antimicrobial efficacy value attributed to some plants is beyond belief. Claims of effective therapy for the treatment of dysentery, diarrhea, respiratory disorders, skin diseases, syphilis, fever, leprosy, eye diseases and kidney and urinary disorders by traditional herbalist and revived our curiosity in the scientific exploration of such herbal medications [12-15]. It was estimated that at one time about 10% of all flowering plants on earth have been used by local communities throughout the world but only 1% have scientifically evaluated. Now we have about 120 plant-based drugs from just 95 plant species which are prescribed worldwide. The pharmaceutical potential of only 5000 medicinal plants are scientifically evaluated from approximately 250,000 species. The current medicines and antibiotics showed a steady decline in treating infectious diseases and pose a great problem due to significant increase in the incidence of bacterial resistance to several antibiotics . The work on new antimicrobial compounds due to increased resistance of many microorganisms towards established antibiotics, has become desirable. Traditional herbs are basic health needs in the developing countries and there are many published researches on the efficacy of herbs against Gram-positive and Gram-negative microorganisms.
One possible approach is to screen/unexplored Indian medicinal bioactive plants extracts for their potential to be used against multiresistant bacteria. India has one of the world’s richest flora with about 120 families of plants comprising 1,30,000 species and about 119 secondary plant metabolites are used globally as drugs. The WHO reported that 80% of world population rely chiefly on traditional medicines/herbs for primary healthcare have steadily increased worldwide in the recent years. Keeping in view this study is designed to evaluate the antimicrobial potentials of Hemidesmus indicus and their bioactive phytochemicals.
Collection of plant materials
The roots of Hemidesmus indicus were obtained from the Himalaya Drug Company Dehradun India. The collected plant material was identified by the department of Pharmacognosy, The Himalaya Drug Company Dehradun. Roots were washed with potable water 2-3 times and once with sterile distilled water and then dried, a homogenous fine powder was made and stored in air tight container till further use.
Preparation of solvent root extraction
For the preparation of the plant extract, the modified method of Alade and Irobi  was used. The powdered root (25 g) were soaked in 100 ml each of the organic solvents (Hexane, methanol) and water in separate flasks and kept on rotating shaker for 72 hours, filtered using Whatman filter paper No.1. The extracts were concentrated to half its volume using rotary evaporator while water extract was concentrated on water bath.
Soya bean casein digest agar/broth of Hi Media Pvt. Ltd., Bombay, India were used for this study.
The Soya bean casein digest Agar were inoculated with approximately 105 CFU/ml of 4 h growth that was incubated at 37°C in Soya bean casein digest broth.
Isolation of volatile bioactive component of H. indicus through clevenger apparatus
The method of Peyson and Richard  was used with little modification. The dry root (1 Kg) was taken in a round bottom flask of a Clevenger type apparatus for 4 hr. at 100°C. The distillate obtained was washed with diethyl ether and dried over anhydrous sodium sulphate. After filtration, the yield of essential was 1.2 g (0.12% w/w) and then it was stored in an airtight glass vessel at 4°C until required.
GC analysis of essential oil
The essential oil of Hemidesmus indicus was analyzed by GC/ MS technique using an GCMS-QP2010 Ultra (Shimadzu Company) GC system, Mass Spectrometer for composition. The identification of volatile oil component was done by comparison of their spectra with NIST 11 lib. /Wiley 8 lib. library data of the GC-MS system and compared their retention indices (IR) with available relevant data. The percentage of peak area relative total peak area represent to relative amount (RA) of each individual component of the essential oil. Determination of RI value of each component relative to the retention time (RT) of series C8-C40 4-alkanes with linear interpolation on the Rtx-5 MS (30 meter × 0.25 i.d. × 0.25 Um film thickness)-column was done.
HPLC analysis of 2hydroxy4-methoxybenzaldehyde (2H4MB)
The fine powder of the root was poured into a glass vessel containing 75% of ethanol and then was filtered and evaporated. The residue of obtained was mixed with n-butanol and water in the ratio of (2:1) and both the layer of n-butanol and water were separated and evaporated under vacuum. The residues were washed with first with petroleum ether then with methanol. The methanolic extract was concentrated and analyzed using HPLC as per standard method of Shimizu et al.  with slight modifications. The extract was passed through Sartorius RC-membrane syringe filter (0.20 m) and 20 μl of filtrate was used for analysis in the HPLC. Shimadzu HPLC (Model SPD-10A UV-VIS Detector) and supercoil LC-18 column (25 cm × 4.6 mm, 5 m) with mobile phase prepared with water, acetonitrile, and acetic acid in the ratio of (50:50:0.1) was used into performing the chromatography. The flow rate and back pressure was consistently retained at 1.0 ml/ minute and 250 psi respectively. UV detector at 210 nm was used to read the compound. The initial total run time was 20 min but after that it was preferably extended up to 40 min . The results obtained were compared with standard.
The antimicrobial activity of the plant extract and the essential (bioactive compound) were tested individually on G+ve and G-ve bacterial strains. All bacterial test strains were received from IMTECH, Chandigarh, India. The G+ve strain used was Staphylococcus aureus MTCC 737 and G-ve bacterial strains were E. coli MTCC 1687; Pseudomonas aeruginosa MTCC 1688 and Salmonella enteric MTCC 3858 and Candida albicans MTCC 3017.
Evaluation of antimicrobial activity
The method of Perez et al.  was modified. Soya bean casein digest agar plates were inoculated with test cultures in SCD broth. Wells of 8 mm diameter were made on the inoculated plate through cork borer and filled with test samples and blank of distilled water, hexane and methanol and positive control of standard antibiotic was simultaneously used. The plates were kept for incubation at 37°C for 18 h. The antibacterial/anticandidal activity was determined by measuring the diameter of zone of inhibition that was observed. Wells were filled with 0.1 ml of 20 mg/ml concentration of each sample (2 mg/well). Bioactivity was determined by measuring Diameter of Inhibition Zones (DIZ) in mm.
MIC/MBC Determination of Bioactive compound (2H-4- MB)
MTT staining method of Scudiero et al. ; Marshall et al.  and Stevens and Olsen  was used to determine the minimum Inhibitory Concentration (MIC) value which is defined as the lowest concentration of the sample that inhibited growth of microorganisms. MTT could convert to Formazan only by living organisms and a blue color appeared in the well. To determine the Minimum Bactericidal Concentration (MBC) approximately 10 μl of the sample from the minimum Inhibitory Concentration assay was spread onto freshly prepared and sterile LB plates and incubated at 28°C over night. The MBC were taken as the lowest concentration that did not allow bacterial growth on the surface of agar plates.
Determination of cellular toxicity using sheep erythrocytes
Cellular toxicity was determined by using the method described by Xian-guo and Ursula  with minor modifications. The extract was 10-fold serially diluted using phosphate buffered saline, 0.8 ml volume of each dilution was taken in Eppendorf tube. The negative control tube (containing saline only) and a positive control tube (containing tap water) were taken for this study. Freshly prepared sheep erythrocytes were used in each tube with a final volume of 1 ml. The tubes were kept for incubation at 37°C for 30 min and were centrifuged for 5 minutes. All tubes after incubation were check for hemolysis.
The yield of the essential oil obtained by hydro distillation of the root of Hemidesmus indicus was 0.12%. The essential oil was evaluated by GC-MS. 10 compounds were identified. The major compound in the oil was 2-hydroxy-4-methoxybezaldehyde (99.41%) (Figure 1 and Table 1).
|10||35.776||61565||0.01||(-)-5-Oxatricyclo [188.8.131.52(4,6)] Dodecane,12-Tri|
Table 1: Peak Report TIC.
The isolated compound 2-hydroxy 4-methoxy benzaldehyde (2H4MB) from Hemidesmus indicus was also analyzed by HPLC based on their standard retention time 6.598 min. The Hemidesmus indicus root extract was also analyzed through HPLC the results showed almost the same Retention time (Rt) in both root extract (6.881 min) and 2H4MB compound Standard (6.598 min). This revealed the presence of 2H4MB compound in root extract of Hemidesmus indicus (Table 2).
|Sample||Retention Time (min)|
|2H4MB Compound standard||11.03|
Table 2: HPLC analysis Hemidesmus indicus extract and the standard bioactive compound.
The antibacterial and antifungal activities of the root extracts of Hemidesmus indicus and 2-hydroxy-4-methoxybenzaldehyde compound were evaluated against 5 test microorganisms including one G+ve bacteria, three G-ve bacteria ads one fungi. Their potency were assessed by diameter of zone of inhibition and MIC/MBC values. Among all the tested extracts hexane extract was found to have maximum zone of 22 mm against Staphylococcus aureus Plate 1 (Table 3, Figure 2) followed by E. coli (18 mm), Candida albicans (18 mm), Pseudomonas aeruginosa (16 mm) and Salmonella enteric (15 mm). The isolated bioactive compound 2H4MB showed the highest diameter of zone of inhibition of 23 mm against Staphylococcus aureus followed by E. coli 16 mm (Figure 3 and Table 3). The Pure compound 2H4MB was also compared with the isolated compound Plate 2-4 as depicted in Table 3 .
|S No||Bacterial/Fungal strains||Inhibition zone diameter (mm)|
|Hexane extract||Methanol extract||Aqueous extract||Isolated Bioactive Compound (IBC) 50 mg/ml||Reference Compound (RC) 50 mg/ml||+ve Control Ciprofloxacin 30 µg/ml|
|1.0.||Staphylococcus aureus MTCC 737||22||16||NAD||23||27||25|
|2.0.||E. coli MTCC 1687||18||14||NAD||16||17||21|
|3.0.||Pseudomonas aeruginosa MTCC 1688||16||12||NAD||14||15||22|
|4.0.||Salmonella enteric MTCC 3858||15||13||NAD||16||17||21|
|5.0.||Candida albicans MTCC 3017||18||16||NAD||20||20||----|
Table 3: Antibacterial and antifungal activity of Hemidesmus indicus root extract and isolated compounds (2H-4-MB).
The MIC values of the Bioactive compound 2H4MB on test microorganisms ranged from 80 μg/ml to 250 μg/ml, MBC values from 150 μg/ml to 250 μg/ml. The MFC values and MIC values of 2H4MB on Candida albicans were 200 μg/ml and 150 μg/ml respectively (Table 4, Figures 4 and 5).
|S. No.||Test microorganisms||2-Hydroxy-4-methoxy-benzaldehyde(µg/ml)|
|1.0.||Staphylococcus aureus MTCC 737||150||80|
|2.0.||E.coli MTCC 1687||200||200|
|3.0.||Pseudomonas aeruginosa MTCC 1688||250||250|
|4.0.||Salmonella enterica MTCC 3858||200||150|
|5.0.||Candida albicansMTCC 3017||200||150|
*Minimum Bactericidal Concentration; **Minimum Fungicidal Concentration; ***Minimum Inhibitory Concentration
Table 4: MIC of 2-hydroxy-4-methoxy-benzaldehyde.
The significant antimicrobial effect of Hemidesmus indicus against all the pathogen confirmed that the compound present in the crude extract are responsible for the effective antimicrobial activity.
The traditional uses of Hemidesmus indicus appeared to have a fairly good degree of correlation with their antimicrobial activity. The cellular toxicity of the alcoholic extract was examined against sheep erythrocytes. Hemolysis of the erythrocytes was not observed at any dilutions of extract ranging from 1:1 to 1:1000. Only the positive control exhibited the strong hemolysis. Whereas the negative control containing only phosphate buffered saline exhibited no hemolysis (Table 5).
|Concentration of sample extracts (200 mg/ml)||Hemolysis|
Table 5: Cellular toxicity testing of plant extracts against fresh sheep erythrocytes.
The herb Hemidesmus indicus showed broad spectrum of action and is non-cytotoxic, it seems to have a promising application in modern medicine as antiseptics and disinfectant. The antibacterial activities of the herb are particularly noteworthy, considering the importance of these organisms in nosocomial infections.