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juniperpublishersjcmah · 5 years ago

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Antioxidant, Antimicrobial And Wound Healing Activity Of Salvadora persica Twig Extracts- Juniper Publishers

Abstract

Wound healing is a complex multifactorial process that results in the contraction and closure of the wound and restoration of a functional barrier. Salvadora persica, commonly known as Miswak was found to contain constituents such as tannins, saponins, flavonoids and sterols. Hence it is thought to evaluate wound healing activity of Salvadora persica since phytoconstituents like tannins, saponins and flavonoids are known to promote the wound healing process due to their antioxidant and antimicrobial activities. Antimicrobial and antioxidant activities of Salvadora persica extracts were studied to understand mechanism of wound healing process. Total phenolic content (TPC) was estimated to screen the prepared extracts by using Folin-Ciocalteu phenol reagent method. Methanol extract showing highest TPC was undertaken for detailed antioxidant, antimicrobial and wound healing activities. Methanol extract showed moderate antioxidant activity on scavenging DPPH, ABTS radicals and by pyrogallol red bleaching method. Methanol extract also showed antimicrobial activity against wound pathogens by agar diffusion method. Methanol extract was formulated into gel and wound healing activity of gel was evaluated using incision and excision wound models in rats. Topical application of prepared gel on the excision wound in rats caused higher rate of contraction and reduced the period of epithelialization when compared to control group animals. In incision wound model, breaking strength of animals treated with the gel containing methanol extract of Miswak twig was found to be significantly (p < 0.001) higher as compared to the control group animals. Hence, the present study revealed that gel containing methanol extract of Miswak twig possess wound healing activity.

Keywords:Incision wound; Excision wound; Antioxidant; Antimicrobial activity; Phytomedicines; Cholesterol plasma levels; DPPH; Ascorbic acid

Abbrevations: TPC: Total Phenolic Content; DPPH: 1,1-Diphenyl-2-picryl-hydrazy; AA: Ascorbic Acid; IAEC: Institutional Animal Ethic Committee

Introduction

Wound healing is an interaction of complex cascade of cellular and biochemical actions healing to the restoration of structural and functional integrity with regain of strength of injured tissues [1]. It involves continuous cell - cell interaction and cell matrix interactions that allow the process to proceed in different overlapping phases including inflammation, wound contraction , re epithelialization, tissue remodeling and formation of granulation tissue with angiogenesis [2]. These events are regulated by several mediators including platelets, inflammatory cells, cytokines and growth factors, and matrix metalloproteinases and their inhibitors [3].

Several factors such as bacterial infection, oxidative stress, necrotic tissue and interference with blood supply, lymphatic blockage and disease condition such as diabetes mellitus delay or reduce the wound healing process. Generally, if the above factors could be altered by any agent, an increased healing rate could be achieved [4].

Nature has gifted us with many herbs having mystical healing properties that are used widely in number of ailments. The use of herbs and medicinal plants as the first medicine is a universal phenomenon. Today, as much as 80% of the world’s population depends on traditional medicine as primary health care needs [5]. Many Plants and their extracts being antioxidant and/or antimicrobial actions have immense potential for the management and treatment of wounds. The phytomedicines for wound healing are not only cheap, well tolerated and affordable but are also purportedly effective and safe as hyper sensitive reactions are rarely encountered with the use of these agents [6]. These natural agents induce healing and regeneration of the lost tissue by multiple mechanisms. Herbal medicines in wound management also involve disinfections, debridement and provide a moist environment to encourage the establishment of the suitable environment for natural healing processes [7].

Salvadora persica (family Salvadoraceae) is an upright evergreen small tree or shrub. It is commonly known as Miswak or Tooth brush tree and is widely distributed in India, Africa, Saudi Arabia, Iran, Israel and Pakistan. It has been claimed in traditional literature to be valuable against a wide variety of diseases [8].

The traditional medicinal use of Salvadora persica as antimicrobial toothbrush stick for oral hygiene, and to treat gum inflammation, is a centuries old practice and a part of Greeko- Arab system of medicine [9]. Pharmacological studies indicated that Salvadora persica L. plant possess anti-microbial, anti-plaque, aphrodisiac, alexiteric, analgesic, anti-inflammatory, anti-pyretic, astringent, diuretic and bitter stomachic activities. It has great medicinal use in the treatment of nose troubles, piles, scabies, leucoderma, scurvy, gonorrhea, boils and toothache, to treat hook worm infections, venereal diseases, for teeth cleaning, in rheumatism, cough and asthma, to lower cholesterol plasma levels, reestablishment of the components of gastric mucosa, and as a laxative [10]. It contains important phytoconstituents such as vitamin C, salvadorine, salvadourea, alkaloids, trimethylamine, cyanogenic glycosides, tannins, saponins and salts mostly as chlorides [11].

However, there is no previous report on wound-healing activity of Salvadora persica twig in literature. The purpose of the present study was to investigate in vivo wound healing activity of Salvadora persica twig. Since antioxidant and antimicrobial agents play an important role in wound healing process, antioxidant and antimicrobial activities of Salvadora persica twig were carried out to find the mechanism behind wound healing process.

Materials and Method

Materials

Gallic acid, 1,1-Diphenyl-2-picryl-hydrazy (DPPH), ascorbic acid (AA), Folin-Ciocalteu phenol reagent, pyrogallol red, 2,2’azinobis (3- ethylbenthiazoline-6-sulphonic acid) (ABTS), potassium persulphate and all other substances used were obtained from Sigma- Aldrich Co. Ltd. Nutrient agar was obtained from Himedia (Mumbai, India). All chemicals used, including the solvents were of analytical grade.

Plant materials

The twig of Salvadora persica were collected from Malvani area in Malad, Mumbai, India and authenticated by Agharkar Institute, Pune, India.

Methods

Extraction of plant material

Authenticated plant material was further dried in shade, powdered and used for extraction. Extraction was carried out using various solvents such as Petroleum ether (60-800), chloroform, methanol, 50% aqueous alcohol and water. The extracts were concentrated in a rotary evaporator under pressure, were kept in desiccators and used for further studies.

Determination of extractive value

10 gm of powdered material were extracted with 100ml solvent using Soxhlet extraction apparatus. The % yield of each extract was determined.

in vitro antioxidant assay methods

Phenolic compounds could be a major determinant of antioxidant potentials of foods and could therefore be a natural source of antioxidants [12]. Hence total phenolic content of the prepared extracts was determined to screen the bioactive extract.

Determination of total phenolic content (TPC)

The total phenolic content was measured using Folin- Ciocalteau reagent as per procedure described by Singleton et al., with some modifications [13]. Test mixture consists of 1ml of extract solution (0.1 or 1mg/ml), 0.5ml of Folin Ciocalteau reagent and 5ml of distilled water. The mixture was incubated at room temperature for 10min. Then 1.5ml of anhydrous sodium carbonate solution (10% w/v) was added and the final volume was made upto 10ml. The final mixture was allowed to stand at room temperature for 2 hr. The absorbance was measured at 725nm using UV-Vis spectrophotometer. The experiment was carried out in triplicate. Gallic acid was used for preparing the standard curve (10μg/ml to 100μg/ml). The total phenolic content in the plant extract was expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g) of extract.

The extract showing maximum TPC was further used for various in vitro antioxidant assays, antimicrobial activity and in vivo pharmacological activities.

DPPH radical scavenging activity

The free radical scavenging activity of active extract was measured by DPPH using the method of Blios [14]. An aliquot of 1ml of the extract solution in various concentration range was added to 3 ml of 0.1 mM DPPH solution. The decrease in the absorbance was determined at 517 nm after 30 min. The percentage scavenging activity was calculated from [(A0-A1)/A0] × 100, where A0 is the absorbance of the control, and A1 is the absorbance of the extract/ standard. A blank is the absorbance of the control reaction (containing all reagents except the test compound). The % scavenging activity and IC50 value of each extract was calculated for the various concentrations. Ascorbic acid was used as standard antioxidant for comparison.

Peroxynitrite pyrogallol red bleach method

Pyrogallol Red solution (100μM) was prepared in 100mM phosphate buffer, pH 7.4. 1ml of extract solution was added to 2ml of 100μM Pyrogallol Red solution.0.5ml of 200μM/liter peroxynitrite solution was added to the mixture and vortexed immediately. After 15 minutes the absorbance was measured using UV-Vis spectrophotometer at 540nm. The % inhibition of pyrogallol red bleaching was determined using the formula [(A1-A2)/A1] X 100, where A1 is the absorbance in presence of antioxidants and A2 is the absorbance in absence of antioxidants. The IC50 values yielding 50% inhibition of Pyrogallol Red bleaching were estimated. Ascorbic acid was used as standard antioxidant for comparison [15].

ABTS assay

ABTS was dissolved in water to a 7mM concentration. ABTS radical cation (ABTS+.) was produced by reacting ABTS stock solution with 2.45mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12-16 hr. The ABTS+. solution was diluted with a phosphate buffer (2mM, PH 7.4) to achieve an absorbance of 0.8 ± 0.014 at 734nm. Extract solutions were mixed with ABTS+. solution, and after 1 min the absorbance was read using UV-vis spectrophotometer at 734 nm. Phosphate buffer solution was used as a blank. The % radical-scavenging activity of the samples was determined using the formula [(Acontrol-Atest)/ Acontrol] X 100, where Acontrol is the absorbance of the control (ABTS+• solution without test sample) and Atest is the absorbance of the test sample (ABTS+• solution with extract). The IC50 values scavenging 50% of ABTS+. were estimated. Ascorbic acid and Trolox were used as standard antioxidants for comparison [16].

Antimicrobial activity

in vitro antibacterial and antifungal activities of methanol extract of bark of Miswak twig were determined by the agar diffusion method against wound pathogens [17]. Bacteria such as Pseudomonas aeruginosa (NCIM 2200), Staphylococcus aureus (NCIM 5022), Streptococcus pyogenes (NCIM 2608), Clostridium perfringens (NCIM 2677), Escherichia coli (NCIM 2065), Klebsiella pneumonia (NCIM 5082), Klebsiella aerogens (NCIM 2239) and fungal such as Candida albicans (NCIM 3471), Aspergillus niger (NCIM 1196) were used as test organisms. The cultures of organisms were procured from NCL (National Chemical laboratory) Pune, India and tested. The petri plates were prepared by pouring melted nutrient agar inoculated with 16 to 18 hr old culture test organisms in a sterile petri dish. Cups were bored in agar by means of sterile cork borer and were filled with either extract to be tested or standard or control and incubated at 37 0C for 18-20 hours. Mixture of dimethyl sulfoxide and water were used as control. Chloramphenicol was served as standard when efficacy was tested against bacteria while fluconazole was served as standard for fungi. Diameter of each zone of inhibition was measured and compared with standard

In-vivo pharmacological activities

Methanol extract of Salvadora persica twig was formulated into 1.5 % Carbopol 971 P NF gel by using extract (1%), ethanol, propylene glycol, triethanolamine and distilled water. Prepared gel was evaluated for skin irritation and wound healing activities.

Animals

Albino Wistar rats of either sex weighing 180-200g were used for the study. The animals were procured from Haffkine Biopharmaceuticals, Mumbai, India. All animals were housed in polypropylene cages under standard experimental conditions with 26+2 0C ambient temperature and 12 h light-dark cycle. The animals were fed standard pellet diet and were provided water ad libitum. All experimental protocols were approved by the Institutional Animal Ethic Committee (CUSCP/IAEC/10 /2013) of C. U. Shah College of Pharmacy, Santacruz (w), India.

Skin irritation studies

Skin irritation study was conducted on albino rats as per OECD guide lines No. 404 (OECD, 2004) in order to evaluate safety of prepared topical gel [18]. The back of the albino rats was shaved to remove the fur carefully, 24 hours before application of the sample. Prepared topical Carbopol gel containing methanol extract of Salvadora persica twig was applied on the skin patches of albino rats and the site of application in terms of erythema and edema was examined at 24, 48 and 72 hours for changes in any dermal reactions. The irritation index was calculated to assess the irritation potential of the prepared Carbopol gel according to Draize Test [19].

In vivo evaluation of wound healing

Incision and excision wound models were used to evaluate the wound-healing activity of prepared topical Carbopol gel containing methanol extract of Salvadora persica twig.

Grouping of animals

For excision and incision wound study, male Wister rats (160- 180g) were selected and were divided into four groups of six animals each. Rats were anesthetized with sodium pentobarbitone injection (45mg/kg, i.p.) and depilated at the predetermined site before wounding. Animals were divided into four groups of six animals each.

Treatment (Group I): Received with topical application of Carbopol gel containing methanol extract of Salvadora persica twig

Positive control (Group II): Received topical application of standard drug ointment i.e. Betadine

Negative (vehicle) control (Group III): Received with topical application of plain Carbopol gel

Negative control (group IV): Animals were left without any treatment

For both excision and incision wound models, the treatment groups were classified and treated in the same manner.

Excision wound model

An excision wound was inflicted by cutting away approximately 500mm2 full thickness of the predetermined area on the anterior-dorsal side of each rat. Each rat was kept in a separate polypropylene cage and was provided with food and water ad libitum. All the test formulations were applied starting from day 0 till complete epithelialization. Wound-healing property was evaluated by % wound contraction percentage and time of wound closure. The wound area was measured immediately by placing a transparent paper over the wound and tracing it out, area of this impression was calculated using the graph sheet. The same procedure is employed every fourth day and wound contraction was expressed as percentage of contraction. The period of epithelialization was calculated as the number of days required for falling off of the dead tissue remnants without any residual raw wound [20].

Incision wound model

A paravertebral long incision of 6 cm length were made through the skin and cutaneous muscle.

After the incision was made, the two ends of the wound were closed with interrupted sutures with stitches 1cm apart using sterile surgical thread and a curved needle. Carbopol gel containing methanol extract of Salvadora persica twig, plain Carbopol gel and Betadine were applied for 9 days. The sutures were then removed on the 8th post - wounding day and the breaking strength of 10-day old wound was measured by tensiometer [21].

Statistical analysis

Results were expressed as means ± SEM (Standard Error of The Mean). Comparisons between groups were performed using one way ANOVA followed by Turkey’s pair-wise comparison test on Graph Pad Instat 3 statistical software.

Results and Discussions

Extraction

The extraction process yielded 1.062 % w/w of petroleum ether extract, 2.24 % w/w of chloroform extract, 17.07 % w/w of the methanol extract, 16.09 w/w % of the of water extract and 5.18 % w/w of 50% aqueous alcoholic extract (Table 1 & 2).

in vitro antioxidant assay methods

Determination of Total Phenolic Content (TPC): The estimation of total phenolic content of the different extracts revealed a high phenol content in the methanol extract i.e. 125.6±0.7mg/g gallic acid equivalent (GAE) followed by 50% aqueous alcohol extract (55.3 ± 0.8752mg/g GAE), water extract (36.33±1.053mg/g GAE), chloroform extract (16.35 ± 1.132mg/g GAE ) and petroleum ether extract (13.1 ± 2.504mg/g GAE) by reference to standard curve (y=0.011x+0.011and r2=0.998) (Table 1).

DPPH radical Scavenging activity: Anti-oxidant activity of Miswak twig was studied by inhibition the stable free radical DPPH. The amount of extract/standard needed for 50% inhibition (IC50). Methanol extract of Salvadora persica twig showed DPPH scavenging activity at higher IC50 value of 63.88 μg/ml as compared to standard ascorbic acid (10.99 μg/ml).

Assessment of pyrogallol red bleaching by peroxynitrite: The plant extract and standard exhibited inhibition of bleaching by Pyrogallol Red method indicating peroxy nitrite scavenging activity. Standard ascorbic acid was able to inhibit bleaching of Pyrogallol Red at IC50 value of 38.08μg/ml. However methanol extract of Salvadora persica twig showed less inhibitory activity with IC50 value of 783.48μg/ml as compared to standard ascorbic acid.

ABTS scavenging assay: Standard ascorbic acid was able to scavenge ABTS radical at IC50 values of 7.23μg/ml. Methanol extract of Salvadora persica twig exhibited moderate free radical scavenging activity by ABTS method with IC50 values of 108.24μg/ ml.

In the present study antioxidant activity levels were found to be relatively high in the methanolic extract of Miswak twig by DPPH, ABTS and Pyrogallol bleaching method; hence oxidative stress may be reduced which is associated with impaired or delayed wound healing process (Table 3).

Antimicrobial activity

The methanol extract from Salvadora persica twig has shown inhibition effects on the growth of all the organisms tested. Amongst the test organisms used, Clostridium perfringens, Candida albicans, Pseudomonas aeroginosa, Staphylococcus aureus were found to be most sensitive to methanol extract of a Salvadora persica twig followed by Klebsiella pneumoniae, Aspergillus niger, Streptococcus pyogenes, Escherichia coli, Klebsiella aerogens. Microbial infection of wounds delays healing and causes a more pronounced acute inflammatory reaction which can lead to further tissue injury and damage. The antimicrobial activity of the extract on wound pathogens partly contribute to the wound healing effect by eliminating infection thus allowing the natural tissue repair processes to start. Hence the results of this study confirm that the herbs possess anti-bacterial activity and this will help keep the wound area sterile, thus promoting wound healing. This fact supports a faster wound healing in the treated groups compared with the control group (Table 4).

In vivo pharmacological activities

Skin irritation study: Carbopol gels containing methanol extract of Salvadora persica twig showed no erythema or oedema on intact rat skin. The primary skin irritation index of the gels was calculated as 0.00. The results indicated that all Carbopol gels did not cause any skin reaction after examining at 24, 48 and 72 hrs. Since the primary skin irritation index of the creams was calculated as 0.00, it can be classified as non-irritant and were found to be safe for topical application.

Excision Wound Study: Table 5 records the reduction of wound area of the different groups over the period of 24 days. It was seen that the faster healing of wound took place in case of animals, which received Carbopol gel containing Miswak extract of Salavadora persica twig and standard. The least rate of wound healing was seen in control group (no treatment) and vehicle control group which received plain Carbopol gel (without extract). A very rapid closure of the wound in the both Carbopol gel containing Miswak extract of Salavadora persica twig and standard treated groups observed between 4 and 8 days of post surgery. After day 8 of post surgery, wound closure was gradual till the total closure of the wound. Total wound closure was observed by the 22 day of post surgery in Carbopol gel containing methanol extract of Salavadora persica twig and by 25 day in control group. On 16th day, wound contraction of standard group was found to be significant (p < 0.001) in comparison to control group. The period of epithelization of standard group (16 days) was also found to be significantly (p<0.001) low as compared to control group (25 days).

Incision wound model: Table 6 compares the tensile strength of the healing skin treated with different gels measured on 10th days. The results of the incision wound healing studies are presented as mean weight in gram ± SD required to break open the resutured wound. The animals treated with methanolic extract and standard showed significant (p<0.001) increase in breaking strength (607.33±8.07gm and 614.33±8.80 respectively) as compared to the control group animals (406.50±9.16gm). This observation confirms that the methanol extract of Salavadora persica twig possesses excellent wound healing property so far as tensile strength of wound healing tissue is concerned.

Conclusion

Results obtained in the present study have shown the antioxidant and antimicrobial activity of methanol extract of Miswak twig. Thus, the external application of methanol extract of Salvadora persica twig on the wound prevented the microbes to invade through the wound, resulting protection of wound against the infections of the various microorganisms. At the same time, external application of the extract entrapped the free radicals liberated from the wound surrounding cells, which are having inherent machinery to protect the cells from the microbes. The faster rate of wound closer in excision wound model indicates the better efficacy of medication the increase in tensile strength of wounded skin indicates the promotion of collagen fibers. The increased tensile strength reveals that the disrupted surfaces are firmly knit by collagen. The synergistic effect of both antimicrobial and antioxidant activity, increased wound contraction and breaking strength accelerated the wound-healing process. Hence, present study confirms the promising wound healing activity of Salvadora persica twig.

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anilv89-blog · 6 years ago

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Solvent Extraction Plants Product Information : SES Agrotech Project Engineers Private Limited

SES AGROTECH PROJECT ENGINEERS PRIVATE LIMITED is a Mumbai based company working towards excellence in the Factory & Manufacturing space, and believe in pursuing business through innovation and technology. Our team comes with several years of industry experience, and comprise of a highly motivated set of specialists & industry experts. Our goal is to be leader in the industry by providing enhanced products, services, relationship and profitability. Our vision is to provide quality products & services that exceed the expectations of our esteemed customers. We firmly believe that our customers are the reason for our existence, and greatly respect the trust that they place in us. We grow through creativity and innovation. We integrate honesty, integrity and business ethics into all aspects of our business functioning. Our mission is to build long term relationships with our customers. We strive towards delighting our customers at every opportunity through exceptional customer service. Our future looks bright as we continue developing a strong base of key customers and increasing the assets and investments of the company. WHAT WE DO SES AGROTECH PROJECT ENGINEERS PRIVATE LIMITED is involved in Manufacture of machinery chiefly employed by grain milling industry and for the oil mills, machinery to clean , sort or grade seed, grain or dried leguminous vegetables; machinery used to produce flour, meal or other ground products; machinery for the extraction or preparation of animal or fixed vegetable fats and oils.

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bhksonar · 5 years ago

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#INFUSIONS! What is an infusion? How does it work with gelato? How can we do it? An infusion is the process of extracting flavors from plant material in a solvent, affecting with that flavor the whole mix (that in our case is the solvent). We can do two different kind of infusions: #COLD or #HOT. - The Cold Infusion is the longest: it needs 6/12/24 hours to be ready. But it is perfect if we want to maintain the freshness of a specific ingredient. Cold infusion is recommended with fresh herbs (basil, mint, ...), flowers (roses, ...) and fresh spices in general (red peppers, ...). - The Hot Infusion, on the other hand, is quite fast: it takes 10/25 minutes. It is the best way to extract the flavors from dry ingredients and spices or to speed up as much as possible the process. In general, the final result is more intense than a cold process, especially with some ingredients such as coffee, vanilla, cinnamon, etc... In our #Compacta there a specific tool used to make infusions during #pasteurization. It is a great component, that you can enjoy making particular combinations and experiments. Regards @bholakumar_thecbiman (at Mumbai, Maharashtra) https://www.instagram.com/p/B_jv9Hqg6kV/?igshid=l1vmwzu13ezv

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journalmaterialscience-blog · 5 years ago

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Synthesis of Calophyllum inophyllum Esters as Biofuel Feed stock Abstract Degrading fossil fuel resources have thrown world into chaos, besides various pollutions and greenhouse gases are a major concern. The fossil fuel shortage in near future is inevitable. This situation triggered the awareness to find alternative and sustainable energy resources. Biofuels are renewable fuel resources developed to replace conventional petroleum based fuels for transportation sector without altering the engines. In the present work, Biofuel is produced from Tamanu seeds (Calophyllum) which is available plenty in GITAM University. The fatty acid rich oil is converted to various esters using methanol, ethanol, 1-propanol, 2-propanol, 1-butanol. These esters were characterized by IR, JHNMR. The Physico- Chemical properties reveal that these esters can be one of the alternatives for the existing fossil fuel. Keywords: Calophyllum inophyllum oil; Fatty acids; Esters; Bio fuel. Go to Introduction Ever increasing petroleum products prices and uncertainties concerning their availability have increased the importance of vegetable oil based biofuel tremendously. The type of vegetable oils used for biofuel production is the parameter that contributes to the cost of the fuel preparation. In USA, mainly soybean oil is used as it is produced in surplus to the edible consumption. On the other hand, in Europe rapeseed oil or sunflower oil or used frying oils are utilized. In Asian countries like Malaysia and Indonesia, palm oil based biofuel are prepared as they have huge surplus of palm oil. India, being one of the major importers of vegetable oils every year, cannot afford to use any edible oil for the preparation of biofuel as almost half of its edible oil consumption depends on imports (9 million tons of edible oil). However, India, due to its tropical climatic conditions and vast terrain region has more than 100 types of trees yielding oil which are yet to be explored. The tree borne oils can be exploited for the preparation of biofuel. Calophyllum inophyllum is a species of family Guttifereae (Clusiaceae), it is a broad leaved evergreen tree widely planted throughout the tropics and easily grown along the coastal area. The tree is valued for its hardiness and beauty as an ornamental tree. Oil from the nuts has been traditionally used for medicine and cosmetics. Annual yield of Calophyllum is around 20-100kg/tree of whole fruits. Trees begin to bear sinificantly after 4-5 years. The nut kernel contains 50- 70% oil. Adeyeye et al. [1], Chavan et al. [2] has reported that the Calophyllum oil exhibited good physico chemical properties and could be used as a biodiesel feedstock and as an industrial application. Atabani et al. [3] has reported that the efficiency of engines has increased when fatty acid blends of Calophyllum biodiesel were used. Sanjid et al. [4], has reported that the Impact of palm, mustard, waste cooking oil and Calophyllum Inophyllum biofuels on performance and emission of CI engines. Krishnaji et al. [5] has reported that the CI engine performance is improved with significant reduction in emissions for the Calophyllum inophyllum Biodiesel without any engine modification. Ravi et al. [6] has studied Performance, Combustion and Emission Characteristics on Single Cylinder Diesel Engine using Calophyllum inophyllum Oil. Sanjay et al. [7] has reported that the tamanu seed crush is directly converted into biodiesel with in-situ trans esterification of fatty acid to methyl and ethyl esters. These components of biodiesel were analyzed by GCMS technique. Mohan et al. [8] has reported that the tamanu oil is a promising alternative fuel for direct-injection in four-stroke VCR engine. Atabani et al. [9], has studied the Fuel Properties of Croton megalo carpus, Calophyllum inophyllum, and Cocos nucifera (coconut) Methyl Esters and their Performance in a Multi cylinder Diesel Engine. Calophyllum is widely grown in GITAM University Figure 1 and in this study we report the synthesis of different ester derivatives of Calophyllum fatty acid from Calophyllum oil to study the effect of chain length on the biofuel properties of the products. Click here to view Large Figure 1 Go to Materials And Methods Calophyllum seeds were collected from GITAM University, Visakhapatnam. Hydrochloric acid, 1-propanol were purchased from MERK life science pvt Ltd., Mumbai. Methanol was purchased from Himedia laboratories pvt. Ltd., Dombivli. Ethyl acetate, Hexane and basic alumina from Finar chemicals Ltd, Ahmadabad. Sodium sulphate, Sodium hydrogen carbonate, Sodium hydroxide was purchased from Fisher scientific pvt Ltd Mumbai. Ethanol was purchased from Changshu Hongsheng fine chemicals from Changshu city Jiangsu Province. pTSA (Para toluene sulphonic acid) was purchased from avra labs, Hyderabad, India. Methods Analytical methods: The GC analysis was performed with an Agilent 6890 N series Gas Chromatography equipped with a flame ionization detector. 1HNMR spectra were recorded on Avance 300MHz in CDCl3. Chemical shift values relative to TMS as internal standards were given 5 values in ppm. IR spectra were obtained on a 1600 FT-IR Perkin-Elmer spectrometer (Norwalk, CT) with a liquid flim between the NaCl. Physicochemical properties namely Free Fatty Acid, density, moisture content, viscosity, flash and fire, cloud and pour and copper strip corrosion were determined using standard AOCS and ASTM methods. A typical procedure for the extraction of Calophyllum oil: The seeds were dried and the kernels were finely powdered and the powder was soxhlet extracted using hexane as a solvent. The hexane was removed under vacuum and the crude oil (40%) was dried under reduced pressure. The crude oil is used without further purification. A typical procedure for the preparation of Calophyllum fatty acid: Calophyllum oil (200gm) and aqueous NaOH solution (36gm dissolved in 450ml distilled water) were stirred mechanically The reaction was monitored by TLC using the solvent system hexane/ethyl acetate (90:10v/v). The reaction was continued until the disappearance of Triglyceride in TLC. After completion of the reaction, the reaction mixture was cooled to 50 °C and acidified with concentrated HCl. The product was extracted with ethyl acetate and washed with water to remove excess HCl. The organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to obtain 180gm of product (78% yields). The fatty acid thus obtained is semi solid. Fatty acid composition of the Calophyllum oil: Fatty acid methyl esters of the Calophyllum oil were prepared by refluxing crude oil with 2% H2SO4 in methanol for 4h. The esters were extracted with ethyl acetate, passed over anhydrous Na2SO4 and concentrated under vacuum. The obtained methyl ester was analyzed for its fatty acid composition by gas chromatography. The fatty acid composition of Calophyllum oil was tabulated in Table 2. A Typical procedure for the preparation of Calophyllum Fatty alkyl esters: Calophyllum fatty acids (50gm, 0.178mol), 1-butanol (26.45gm, 0.356mol), p-TSA (0.5gm based on 1% weight of fatty acid) and 50ml of toluene were collected in three necked round bottomed flask equipped with a thermometer, condenser and Dean stark apparatus. The reaction mixture was stirred at 130-140 °C. The reaction was monitored by checking TLC using hexane: ethyl acetate (90:10v/v). After the disappearance of fatty acid and the theoretical amount of water was collected. The reaction mixture was cooled to room temperature. The crude was concentrated under vacuum to remove alcohol and toluene. The resultant product was dissolved in hexane, washed with NaHCO3 solution, passed over anhydrous Na2SO4 and dried under vacuum to afford alkyl esters of Calophyllum fatty acids. The unreacted fatty acid was removed by passing the crude over basic alumina. The ester product was analyzed for acid value. The esters were characterized by IR, 1HNMR Spectral studies. Butyl ester of Calophyllum fatty acid IR (neat, cm-1): 3007(-C=C-H); 1740(-C=0); 1172(C-O). 1HNMR (CDCl3, δ-ppm): 0.85-0.92(t, CH3), 1.2-1.38 & 1.56- 1.67(m, -CH2), 2.0-2.1 (m, CH2CH=CH2), 2.29-2.31 (t, CH2C=O), 3.93-4.01 (t, CH2O), 5.45 (m, -CH=CH). The above procedure was followed to prepare various alkyl esters using different alcohols (methanol, ethanol, 1 & 2-propanol) with Calophyllum fatty acids. And those esters were characterized by IR & 1HNMR studies. Methyl ester of Calophyllum fatty acid IR (neat, cm-1): 3007(-C=C-H); 1742(-C=O); 1118(C-O). 1HNMR (CDCl3, δ-ppm): 0.90(t, CH3), 1.2-1.38 & 1.52- 1.65(m, -CH2), 2.0-2.1 (m, CH2CH=CH2), 2.29-2.31 (t, CH2C=O), 3.68 (t, CH2O), 5.38(m, -CH=CH). Ethyl ester of Calophyllum fatty acid 1HNMR (CDC13, δ-ppm): 0.89(t, CH3), 1.28-1.37(m, -CH2), 1.65-1.7 (m, CH2CH=CH2), 2.25-2.35 (t, C H2C=O), 3.7 (t, CH2O), 4.15 & 4.30, 5.4(m, -CH=CH). 1-propyl ester of Calophyllum fatty acid 1H NMR (CDCl3, 5-ppm): 0.82-0.90(t, CH3), 1.2-1.4 & 1.601.72 (m, -CH2), 2.0-2.1 (m, CH2CH=CH2), 2.30-2.35 (t, CH2C=O), 3.68 (t, CH2O), 4.15 & 4.26, 5.35 (m, -CH=CH). Physico-chemical properties: various physic-chemical properties studied are a. Kinematic viscosity determination by ASTM D445: b. Biofuel, the most important physico chemical property is kinematic viscosity. Since it affects the operation of fuel injection equipment. Results show that there is change in viscosity from the oil into the various esters. c. Flash point: It is the lowest temperature at which a liquid forms an ignitable mixture in air near the surface of the liquid. Flash point is measured by ASTM D93 method. The results are in agreement with ASTM D93, which requires that flash point to be minimum 93oC of the synthesized esters. d. Pour point: The pour point indicates the ability of the liquid to flow at lower temperatures. The pour points were determined by ASTM D97 method using pour point apparatus manufactured by Cultures Instruments India LLP, Bangalore, India. All the samples were run in duplicates. e. Cloud point: The cloud point is indicates the ability of the liquid to start clouding at which temperature. The cloud point was determined by ASTM D2 500 using cloud point apparatus manufactured by Cultures Instruments India LLP, Bangalore, India. f. Copper strip corrosion: Determines the corrosiveness of the substance As per ASTM D 130 method. Copper strip corrosion was determined by using copper strip corrosion bath which was purchased from Cultures Instruments LLP, Bangalore, India. A polished copper strip in immersed in 30ml of sample being tested at 50 °C for 3h. After 3 h the Cu strip is removed, washed and the colour and tarnish level was assessed against the ASTM copper strip corrosion standard. g. Density: Density is determined by Pycnometer. The density of Calophyllum esters were measured by ASTM D4052 method at 250 °C. h. Moisture content: Moisture content was calculated by using ASTM D2974-87 method. i. Acid number: Acid number indicates the amount of free acid present in sample. The acid number was calculated by using ASTM D974/01 with help of standard KOH solution. Go to Results And Discussion The advantages of using non edible oils as fuel compared to diesel fuel are liquidity, ready availability, renewability, lower sulphur and aromatic content, biodegradability, higher cetane number These oils are extremely viscous converting these to biofuel decreases its viscosity. Blending of these oils and biofuel with some % of diesel fuel was a suitable method to reduce choking and for extended engine life. The present study is focused on the use of non-edible Calophyllum oil rich in free fatty acid and was widely grown in GITAM University and also along the coastal areas of India. The seeds of Calophyllum were collected dried and crushed. The oil was soxhlet extracted using hexane as a solvent. The extracted crude oil is greenish yellow and it gets darkened during the storage. The oil having disagreeable odour. The physico chemical properties of crude oil are tabulated in Table 1 and were carried out as per (ASTM) -6751. Extracted oil consisted of pure triglyceride and free fatty acids along with minor components. The crude oil was trans esterified using methanol to obtain Calophyllum fatty acid methyl esters and the methyl esters were determined for fatty acid composition using gas chromatography and the results were tabulated in Table 2, Figure 2. Click here to view Large Table 1 Click here to view Large Table 2 Click here to view Large Figure 2 Calophyllum oil was esterified with different alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, using H2SO4 or p-TSA by two step process Figure 3. The first step involving the formation of free fatty acid by base hydrolysis. The fatty acid is a semi solid and the next step involving the esterification of free fatty acid with various alcohols, the ester thus obtained was liquid having a pleasant odour and the esters of Calophyllum fatty acids were characterized by NMR and IR spectral studies. The presence of peak in IR around 1740cm-1(- C=O) and 1174(C-O-C) and the presence of peaks in NMR around 2.29-2.31(2H, t, CH2C=O), 3.93-4.01(2H, t, CH2 the formation of ester was given below in Figure 4. (A-methyl, B-ethyl, C-1-propyl, and D-1-butyl) Click here to view Large Figure 3 Click here to view Large Figure 4 Click here to view Large Table 3 The synthesized alkyl esters of Calophyllum fatty acids were evaluated for their physico chemical properties and fuel properties the results were tabulated in Table 3. The kinematics viscosity of the oil at 40 °C and 100 °C are 63.12cst and 10.67cst. While that of esters in the range of 6-22 and 2-9cst. It was found that the flash point of are in agreement with ASTM D93, The flash point of the oil is 185.2 °C. While that of esters are in range of 100-200 °C. The pour point of the oil is -9.8 °C. While that of esters in range of -30 to 0 0C. The cloud point was determined by ASTM D-2500 using cloud point apparatus manufactured by Cultures instruments LLP, Bangalore, India. The pour point of the oil is 90 °C. While that of esters in range of 0 0C to 20 0C. The color and tarnish of copper strip level was assessed against the ASTM copper strip corrosion standard and all the esters exhibited copper corrosion values of 1a. Go to Conclusion There is a tremendous demand for non-edible based oils due to depletion of mineral resources and growing concerns for eco-friendly products. Calophyllum oil, widely grown in coastal area as well as in GITAM University can be used as a feedstock for bio-fuel production. In the present study methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, esters were prepared from corresponding alcohols and are evaluated for fuel properties. The fuel properties were compared with literature and ASTM D6757 biodiesel standards. The study revealed that various esters of Calophyllum oil seem to be a satisfactory feed stock for future biodiesel production. For more Open Access Journals in Juniper Publishers please click on: https://juniperpublishers.com/ for more details click on the juniper publishers material science

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