|
 
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| ORIGINAL ARTICLE |
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| Year : 2018 | Volume
: 3
| Issue : 1 | Page : 18-26 |
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Antimicrobial effects of leaves of Indian herbal plants with reference to peptic ulcer
Mayank Kulshreshtha1, Harinath Dwivedi2, Manjul Pratap Singh2
1 Department of Pharmacology, School of Pharmacy, Babu Banarasi Das University, Lucknow, Uttar Pradesh, India
2 Department of Pharmaceutics, School of Pharmacy, Babu Banarasi Das University, Lucknow, Uttar Pradesh, India
| Date of Submission | 07-Oct-2017 |
| Date of Acceptance | 01-Feb-2018 |
| Date of Web Publication | 11-Apr-2018 |
Correspondence Address:
Prof. Mayank Kulshreshtha
Department of Pharmacology, School of Pharmacy, Babu Banarasi Das University, Lucknow, Uttar Pradesh
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ed.ed_18_17
Objective: Gastrointestinal (GI) diseases affect a large part of the population. Peptic ulcer (PU) which is very common disease affects the stomach and duodenum. Many microbial species such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus tubingensis etc are responsible for PU directly or indirectly.
Materials and Methods: The aim of this study was to find out the effects of aqueous and ethanolic extracts of leaves of Quisqualis indica, Elaeocarpus ganitrus and Prosopis cineraria on PU-associated microorganism. Minimum inhibitory concentration (MIC) and the synergistic effects of selected plant extracts with renowned standards (ciprofloxacin, norfloxacin, ketoconazole, and fluconazole) were estimated. Disc diffusion and solid dilution methods were used for the determination of antimicrobial effects and MIC.
Results: Studies revealed that the plant extracts were highly effective against selected microbes. Escherichia coli was found to be the least affected microbial species. Different infectious diseases of GI tract might be cured using formulations having these selected plant extracts. The zone of inhibitions (ZOI) was observed ranging from 2.3 ± 0.57 to 12.3 ± 0.59 mm. The maximum ZOI was observed (12.3 ± 0.59 mm) for ethanolic extract of Elaeocarpus ganitrus against Pseudomonas aeruginosa.
Conclusion: The herbal extracts were found to be highly promising against selected microbial species associated with PU.
Keywords: Elaeocarpus ganitrus, Prosopis cineraria, Quisqualis indica, gastrointestinal diseases, minimum inhibitory concentration, peptic ulcer
How to cite this article:
Kulshreshtha M, Dwivedi H, Singh MP. Antimicrobial effects of leaves of Indian herbal plants with reference to peptic ulcer. Environ Dis 2018;3:18-26 |
| Introduction | |  |
Peptic ulcer (PU) is an acute or chronic disease which is characterized by an imbalance between the aggressive and defensive factors of the digestive tract.[1] In the United States, every year, PU affects around 500,000 people (about 78% between the age of 25 and 64 years).[2] An estimated annual health-care cost for PU is about $10 billion.[3] The microorganisms accountable for PU like Escherichia More Details coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Bacillus subtilis  (B. subtilis), Staphylococcus aureus (S. aureus), Aspergilus tubingensis ( A. tubingensis) etc. E. coli, a Gram-negative bacterium, is the most pervasive infecting organisms (family – Enterobacteriaceae) and is not harmful for the humans [4] but sometimes causes infections such as urinary tract infections.[5]P. aeruginosa is known as an opportunistic pathogen because it infects especially immunocompromised patients with cystic fibrosis, cancer, or AIDS.[6] It mainly attacks two-third of the hospitalized patients resulting 90% deaths by cystic fibrosis.[7]B. subtilis is ubiquitous in nature and found mainly in soil, in either decomposing plant matter [8] or humane digestive tract.[9]S. aureus is one of the main reasons for hospital- and community-gained infections,[10] which affects the bloodstream, skin, soft tissues, lower respiratory tracts, central venous catheter-associated bacteremia, ventilator-assisted pneumonia, infection of endocardium, and heart valve and bone infection.[11] A. tubingensis causes keratitis (an inflammation of eye).[12]
Prosopis cineraria (P. cineraria) family – Fabaceae is an indigenous plant mentioned in Ayurveda with several clinical benefits.[13] Locally, it is called “Kalpavriksha.”[14],[15] According to Hindu mythology, it is referred as Shami tree because Lord Rama and Laxamana placed their swords and weapons on this tree.[16]P. cineraria is reported to have various pharmacological activities such as analgesic, antipyretic, antihyperglycemic, antioxidant, antihypercholesterolemic, antitumor, nootropic, respiratory, gastrointestinal, anticonvulsant etc. due to the presence of various secondary metabolites.[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28]
Elaeocarpus ganitrus Roxb. (E. ganitrus), family – Elaeocarpaceae is an evergreen tree commonly known as Rudraksha that has hard and highly ornamental stony structure known as bead.[29] Ayurveda refers this as body strengthening canon and can be wear either on wrist, arm, or other parts of the body.[30] It is used in stress, anxiety, depression, palpitation, nerve pain, epilepsy, migraine, lack of concentration, asthma, hypertension, arthritis, and liver problems. On behalf of Ayurvedic medicinal system, abrasion of Rudraksha can have a positive effect on heart and nerves.[31],[32],[33],[34] Various scientific reports are available on various pharmacological activities including anti-inflammatory, antihypertensive, antidepressant, central analgesic, antioxidant, antimicrobial, antimalarial, cytotoxic, antidiabetic, anxiolytic, antiasthmatic etc.[35],[36],[37],[38],[39],[40],[41],[42],[43],[44]
Quisqualis indica Linn. (Q. indica), family – Combretaceae, is a tough hiker, ligneous vine (2.5–8 m), also known as Rangoon creeper. It is indigenous to Africa and Indo-Malaysian region and is cultivated all over India.[45] It is an evergreen plant having vigorous growth needing sturdy support and can get quite out of hand on its sympathetic growing site.[46] The plant requires full sunlight and regular watering to keep the soil moist and needs a base position for the vine to grow on.[47] Leaf, seed, and root extracts are used as an anthelmintic. Juice of leaves eases flatulence. Infusion of leaves is used to treat boils and ulcers (externally).[48],[49] It is generally a fancy plant with proven various pharmacological activities such as anti-inflammatory, antipyretic, immunomodulatory, anti-staphylococcal, acetylcholinesterase inhibitor, antioxidant etc.[50],[51],[52],[53],[54],[55],[56],[57]
The study undertaken is to assess the antimicrobial effect of herbal extract obtained from P. cineraria, E. ganitrus, and Q. indica against different species of microorganisms which may affect the GI tract (GIT) to cause PU and other gastric abnormalities. The literature survey has confessed that there is a lack of scientific data on antimicrobial profile, minimum inhibitory concentration (MIC), and combined effects of plant extracts with different standards against selected microbes. The study undertaken might be useful for the preparation of different herbal formulations to cure PU and protection of GIT from different infectious agents.
| Materials and Methods | | |
Chemicals and reagents
Ciprofloxacin and fluconazole were provided as a free gift sample from Alkem Pharmaceuticals and Sun Pharmaceuticals, respectively. Norfloxacin and ketoconazole were purchased from local market of Lucknow, India. Ethanol was purchased from Changshu Yangyuan Chemical, China. All the chemicals and reagents used were of analytical grade.
Collection and authentication of plant material
Leaves of Q. indica, P. cineraria and E. ganitrus were collected from National Botanical Research Institute, Lucknow, in July 2015 and authenticated by Dr. Sunita Garg, NISCAIR, Delhi (Ref No. NISCAIR/RHMD/2015/2862/55-1, NISCAIR/RHMD/2015/2862/55-2, and NISCAIR/RHMD/2015/2862/55-3, respectively).
Extraction methodology
The extractions of the leaves were carried out according to the official procedures. The plant materials were dried in light-free area and powdered with the help of mechanical grinder. The powder (25.0 g) of the plant material was defatted with petroleum ether (60°C–80°C) using a Soxhlet extractor for 72 h at a temperature not exceeding the boiling point of the solvent. After that, powdered material was dried and treated with different solvents and then conserved in sterile bottles in refrigerated conditions (2°C–4°C) for further use and to explore the antimicrobial potential of various extracts.[58]
Identification of secondary metabolites by phytochemical screening
Different metabolites such as carbohydrate, alkaloids, flavonoids, tannins, proteins, and terpenoids were estimated for their presence in different leaves extracts.[59]
In vitro antimicrobial studies of different plants extract
Antimicrobial studies of different extracts were done by agar well diffusion method. Microbial strains such as Gram-positive bacteria (S. aureus MTCC 1430, B. subtilis MTCC 0441) and Gram-negative bacteria (P. aeruginosa MTCC 2453, E. coli MTCC 1573) and a fungal strain (A. tubingensis MTCC 2546) were purchased from Institute of Microbial Technology, Chandigarh. Culture media for the activation of microbes were prepared according to the official protocol as given in Microbial Type Culture Collection literature. Extracts were dissolved in dimethylsulfoxide (DMSO), whose concentration range was not kept more than 10%. Basic principle of agar well diffusion method is diffusion of the antibiotic from the wells through a solidified agar layer in a Petri dish More Details to an extent such that growth of active microorganisms was inhibited in the zone around the well having solution of the standard agents. The microorganisms were inoculated into sterile molten nutrient agar which has been cooled to 45°C, mixed well, poured into sterile Petri dishes and allowed to solidify. Five wells were made by sterile cork borer. Test samples of concentration 25, 50, and 100 μg/ml by dissolving in DMSO, standard drugs, and control were poured into the corresponding well by micropipettes. Inoculated plates in triplicate and Petri dishes were left at room temperature. To allow the diffusion of the samples, Petri dishes were incubated at corresponding temperature of each organism for 24 h. The diameter of the zones of inhibitions (ZOI) was measured in millimeter.[60]
In vitro antimicrobial studies of different extracts with standards
Different dilutions of extracts were added with known standards and followed by agar well diffusion method. Concentration of herbal extracts and standards mixed together and antimicrobial effect were observed with above-mentioned strains.
Determination of minimum inhibitory concentration by solid dilution method
MIC is the minimum concentration of any compound compulsory for a full inhibition of the microbial growth. In this method, the dilutions of the samples under test were made in agar. The agar had the test samples under subsequently poured onto Petri dishes. Nutrient agar was added; the solution under test and the mixture were poured into sterile Petri dishes and allowed to set in the form of a block. After this, second amount of agar was poured onto the solid and allowed to set with the Petri dishes on the bench. To allow dissolving of the drug, the plates were incubated whole night. The streaking of microorganism was in the direction running from the higher concentration to the lower concentration. The result was calculated by measuring the length of growth of microorganism and the total length of the agar surface streaked; then, if total length of desirable growth was x cm and total length of actual growth was y cm, the minimum inhibitory concentration of compounds was determined using the formula
c × y/x mg/ml
Where c is the final concentration in μg or mg/ml of the drug in the total volume of the medium.[61],[62],[63]
| Results and Discussions | | |
Herbal medicines are stable and the primary form of treating problems of majority of people in well-developing countries. Moreover, as per the availability of western medicines, the number of people using one or another form of complementary medicines is rapidly growing worldwide. Increasing knowledge of physiological process and the effect of herbs on human system has enlarged the application of medicinal plants. According to the report by the World Bank in 1997, it is apparent that the significance of plant-based medicines has been increasing all over the world. Nearly 50% of medicines in the market are made of natural basic materials. Interestingly, the market demands for medicinal herbs are likely to remain high because many of the active ingredients in medicinal plants cannot be yet prepared synthetically.[64]
Epidemiology of PU disease includes the various factors such as environmental factors, Helicobacter pylori (H. pylori) infection, nonsteroidal anti-inflammatory drugs use and smoking. However, these natural factors do not tell the full story of the time trends and the birth–cohort effect for PU disease. In particular, H. pylori was a prevalent human infection well before the late 1800s so that this infection per se cannot explain the rise in ulcer prevalence and shift from gastric ulcer to duodenal ulcer.[65]
Phytochemical screening of different plant extracts
The quantitative parameters and physicochemical properties of the leaves were performed on behalf of Ayurveda Pharmacopoeia of India. Testing of extracts with different reagents shows the presence of alkaloids, glycosides, tannins, flavonoids, fats and oils, carbohydrates, reducing sugar, proteins, saponins and terpenoids [Table 1] respectively. These secondary metabolites are responsible for the cure of various GI diseases. Apart from that, investigations of medicinal plants are getting importance among the scientists both for human health and for food industry.[66],[67],[68],[69],[70],[71],[72],[73],[74],[75] Foods that protect the natural additives have become suitable due to greater consumer knowledge and active regarding synthetic chemical additives. Food industries are highly active for natural preservative with no or little adverse effects and to keep their food preserved for long period of time.[76],[77] | Table 1: Phytochemical analysis of different extracts of Prosopis cineraria, Elaeocarpus ganitrus, and Quisqualis indica
Click here to view |
In vitro antimicrobial studies of different plants extract
[Table 2] shows that ZOI found to be very low in case of E. coli (between 2.3 ± 0.57 and 4.6 ± 0.57), so it is clearly proved that plant extracts do not have major effect on E. coli which is a human-friendly bacterium that can be found in intestinal microflora of a variety of animals including humans; not all the E. coli strains are harmless, but some are responsible for fatal diseases in human as well as in mammals.[78] The maximum ZOI was observed to be 12.3 ± 0.59 mm in case of ethanolic extract of E. ganitrus (EEEG) against P. aeruginosa [Figure 1]. In case of A. tubingensis, maximum ZOI was found to be 9.7 ± 0.41 mm. The different ZOIs for different extracts and standards against various microbes are shown in [Table 2] and [Table 3]. | Figure 1: Effect of ethanolic extract of Eleocarpus ganitrus against Pseudomonas aeruginosa
Click here to view |
In vitro antimicrobial studies of different plants extract with standards
[Table 4] shows the antimicrobial activity of extracts with different standards and it was found that combination of plant extracts and standards has better antimicrobial activity as compared to pure plant extract. On the basis of such results, we can say that the combination has some synergistic effect. Further research works are needed for confirmation of claimed effect. | Table 4: Antimicrobial studies of different plants extract with standards
Click here to view |
Minimum inhibitory concentration of different extracts and standards on different microbial strains
The MICs of different extracts and extracts with standards are shown in [Table 5] and [Table 6]. The minimum MIC found to be 0.22 ± 0.02 μg/ml in case of AEPC against S. aureus. In case of A. tubingensis, effective MIC was found to be 0.23 ± 0.04 μg/ml with EEEG which is some nearest to standard (fluconazole). [Table 6] clearly proves that MICs of extracts with standards are much better compare to alone extract. Hence again, it can be concluded that the formulation of such combination may be beneficial in the future. | Table 5: Minimum inhibitory concentration of different extracts and standards on different microbial strains
Click here to view |
 | Table 6: Minimum inhibitory concentration of different extracts with standards on different microbial strains
Click here to view |
| Conclusion | | |
Since ancient times, plants have been used by several societies to treat number of diseases, including infections. Traditionally mentioned plants play a target-oriented role to cover the basic health needs. The therapeutic importance of such plants is due to various secondary metabolites present in it, which may produce good pharmacological actions on human. In the present study, all three medicinal plants were found to be promising source of secondary metabolites in which the flavonoids are well-known antimicrobials, so the antimicrobial activity of these plants is directly proportional to the amount of the secondary metabolites mentioned above. Extracts are least effective against E. coli which ensured the effectiveness of developed formulation prepared by these extract in the future. It may protect the GIT and related diseases. This scientific data may be helpful to generate the antimicrobial profile and monograph of the plants, to develop the various herbal formulations, and to guide the new scientists in the future. The presence of various metabolites in extracts may play a key role in opening various doors in the treatment of several diseases.
Acknowledgment
I (Kulshreshtha) would like to express my hearty thanks to Dr. Harinath Dwivedi and Dr. Manjul Pratap Singh (Sr. Assistant Professors), Department of Pharmaceutics, School of Pharmacy, BBD University, Lucknow, for providing me research oriented advises; Dr. Sunita Garg, NISCAIR, New Delhi, for the authentication of leaves material; Institute of Microbial Technology, Chandigarh, for providing microbial strains; National Botanical Research Institute, Lucknow, for the collection of leaves material and last but not the least Mr. Karuna Shanker Shukla, Assistant Professor cum Ph.D. Research Scholar, School of Pharmacy, BBD University, Lucknow, for their helping nature and helping me to make this research possible.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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