Analytical techniques involved in extraction and purification of phytochemicals from a medicinal plant
Phytochemicals are naturally occurring chemical compounds that help in the plant defense from competitive species, free radical damage, stress factors, pathogen attack and toxic environmental conditions. They give plants color, ﬂavor and smell and hence are abundantly found in medicinal plants. The aim is to extract and purify phytochemicals as they are known to have signiﬁcant anti-oxidant, anti-microbial, anti-cancerous and anti-inﬂammatory properties; which are indeed the essential qualities that built the emerging aspects of drug design and development. Extraction results in isolation of plant analyte mixtures based on the solvents used. Soxhlet extraction from dried leaves of a medicinal plant using organic solvents of increasing polarity such as hexane, ethyl acetate and methanol, was used as the extraction technique in this analysis. Micro-ﬁltration followed by concentration by rotary evaporation yielded a debris-free thick plant extract. Chromatographic techniques were applied for separation of the compounds. To identify the possible number of compounds present in the extract, TLC (Thin layer chromatography) optimization of crude extract was done with Silica gel 60 F254 plates using increasing solvent polarities. Scaling up with column chromatography was carried out using silica gel 60-120 mesh column to separate the phytochemical mixture by step-wise gradient of ethyl acetate in hexane. About 16 fractions were collected from the column and the compounds eluted were detected using optimized TLC. To purify compounds from the fractions, preparative TLC was performed using silica gel G. For very diﬃcult separations, reverse phase HPLC was employed using C18 column. HPLC parameters and the mobile phase compositions were optimized to yield better resolution of desired compound and the impurities present. Apart from this, HPLC was also used for identiﬁcation of various standard plant compounds (like phenolic acids.) and quantiﬁcation of such compounds in the crude extract using standard calibration curves. The puriﬁed compounds can further be used for medicinal applications.
Keywords: phytochemicals, medicinal plant, Soxhlet extraction, chromatography, puriﬁcation, quantiﬁcation
|4HBA||4-Hydroxy Benzoic Acid|
|AUC||Area Under the Curve|
|HPLC||High performance liquid chromatography|
|PCA||Para coumaric acid|
|PGA||Phenyl glycolic acid|
|RP- HPLC||Reverse phase High Performance Liquid Chromatography|
|TLC||Thin layer chromatography|
Statement of the Problems
Crude extracts from plants have been used to treat various pathological conditions over centuries. They have proven to be quite effective against various pathological conditions and are suitable for long term use. However, the major drawback with such extracts is that their therapeutic effect is slow. The major reasons behind this includes; the concentration of compounds responsible for the specific activity is low, complex reactions among the compounds in the crude mixtures, heavy metal toxicity from the extract preparations, etc. Therefore, current research focuses on identifying the compounds from such extracts that are responsible for the therapeutic effect for developing new drug leads . Hence, the aim of the work was to establish proper analytical techniques required to extract, identify, isolate and purify phytochemicals from the chosen medicinal plant (Harborne, et al, 1980). Also, these techniques could be applied for other applications like extraction and isolation of flavouring agents, natural dyes, cosmetic related compounds and so on. Therefore, in this analysis, the following problems were addressed:
1. Plants have a wide range of compounds with different chemical properties (Altemimi, et al, 2017). This pool of compounds have to be extracted at higher yields without affecting their structural integrity and stability.
2. Identification and isolation of compounds from the extracted crude. Choosing the right chromatographic techniques, conditions and protocol optimizations are essential for obtaining pure fractions.
3. To obtain compounds with high purity. This is very important for any further use of those compounds, in terms of structural characterization and other applications. The purification technique depends on the chemical nature of the desired compound as well as the impurities.
4. Using HPLC for identification and quantification of standard plant phenolic acid in crude extract. Also, this study includes proper optimization of mobile phase for identification and resolution of structurally similar compounds in plants.
Objectives of the Research
1. Extraction of crude extracts from leaves of medicinal plant by Soxhlet extraction using different solvents.
2. Identification, isolation and purification of individual phytochemicals/mixtures using chromatographic techniques.
3. Reverse Phase- HPLC for rapid identification of compounds.
3.1 Designing protocol for resolution of structurally similar phenolic compounds
3.2 Identification and quantification of standard phenolic acid in plant crude extract
1. Developing robust protocols for isolation of important chemical compounds from natural sources, with high yield and purity.
2. Elucidation of structure of purified compounds using various analytical and spectroscopic techniques.
3. Testing applications such as anti-microbial, anti-oxidant (Rahman Gul, 2017), anti-inflammatory, anti- cancer activities depending on the physical and chemical properties of the compounds.
The use of medicinal plants is becoming popular all over the world. The knowledge of traditional medicine were developed through trial and error method over many centuries ( Yuan, et al, 2016). In spite of the modern therapeutic approaches, traditional medicine is still preferred, sometimes together with allopathic treatment (Aakanksha Tiwari, 2018). At present, the main strategy of the scientists is to discover particular active ingredients from such medicinal sources. Some examples of such discoveries include salicylic acid, morphine, aspirin and so on.
Several instruments and methodologies are being developed at present with the helping hand from technological advancements. In order to extract the chemical compounds from natural sources, various techniques are used based on the extraction conditions. These include simple material-solvent incubation (maceration), applying continuous heat (Soxhlet extraction), assisted extraction (ultrasonication , homogenization), Super-critical-fluid extractions and so on ( Azwanida NN, et al ,2015 ). Depending on the physical and chemical properties of the desired compounds, various solvents of appropriate polarity are used. For further separation and isolation of compounds from the extracts, various chromatographic techniques are utilized based on the chemical properties of the analytes. Some of these include HPLC, Gas Chromatography, column chromatography, Preparative / Analytical TLC, Affinity chromatography, Ion exchange chromatography, Gel filtration chromatography and so on. The type of chromatographic technique for separation is determined by the nature of the compound to be isolated. For instance, polar analytes are separated using Reverse Phase-HPLC, enzymes and recombinant antigens are purified using affinity chromatography, size based separation of biomolecules is achieved by gel-filtration chromatography and so on. The advancement in the instrumentation and application of downstream processing have created a significant impact in quick recovery of analytes in its pure and native form, with minimal contaminations and maximum yield.
The present study is focused on important analytical techniques which are involved in extraction and purification process of plant phytochemicals. This study helps in understanding the principles, methodologies, requirements and precautions needed to be taken while carrying out the experiments involved in phytochemical analysis. Extraction was carried out by two methods namely Soxhlet extraction and ultrasonication. The plant debris were removed by filtration followed by concentration using rotary evaporation. Isolation and purification of plant compounds from the obtained extracts was carried out using chromatographic techniques. The compounds were identified using TLC and visualized in iodine chamber. For scale up, silica gel column chromatography was performed for isolation of several fractions of compounds. Purification of each fraction was done using preparative TLC.
Detection and identification of structurally similar standard phenolic acids, namely- 4- hydroxybenzoic acid (4HBA), Phloretic acid (PA), Phenyl glycolic acid (PGA) and para- coumaric acid (PCA) was performed using RP- HPLC. In order to achieve so, proper optimization of mobile phase was carried out. Apart from this, identification and quantification of PCA in the plant crude extract was also performed. While executing these experiments, proper instrument handling and maintenance was learnt.
An extract was obtained when the plant material was subjected to solvent by breaking down plant cells and releasing their contents . Soxhlet extraction is a continuous heat extraction where the sample placed in extractor is subjected to hot solvent vapours. Due to application of heat, the solubility of the phytochemicals is enhanced thereby improving the yield. Recycling of solvent is an added advantage in this method which leads to less solvent wastage and ensures complete extraction of the plant material. Also, other technique of extraction such as ultrasonication was used. Ultrasonication is suitable for extracting compounds from smaller amounts of sample material. The ultrasound waves that are released from the probe causes cell disruption which in turn facilitates release of intracellular plant components into the solvent medium. The advantage of this technique over the others is the stability and integrity of the extracts and thus improving efficiency (Prashant Tiwari ) ( Anup Kumar Das, 2015 ). A debris free extract was obtained by filtration. Depending on the size of the debris, appropriate filters have to be chosen. To obtain solvent free extract, concentration by rotary evaporation was carried out. The rotary evaporator works by increasing the rate of evaporation of the solvent by reducing the pressure thus lowering the solvent boiling point; rotating the sample to increase the effective surface area and heating the solution. These help in faster evaporation of solvent and hence results in quick removal of solvent from the extract. TLC was used to identify the compounds present on the crude extract. A thin layer of silica gel coated onto a flat plate (glass/aluminium) constitutes the stationary phase. The sample loaded on the gel is separated with solvent mixture of varying polarity based on the polarity/partition coefficient of the compounds ( Jim Clark ) The separated compounds can be visualized using UV and staining methods like iodine, ninhydrin and so on. The extract was subjected to silica gel chromatography for large scale isolation. Since silica gel is a polar stationary phase, compounds of increasing polarity gets eluted starting from non- polar ones. The mobile phase is non-polar in nature and the polarity is increased gradually for better separation of compounds. Purification of the fraction obtained from the column chromatography was carried out using preparative TLC. The principle is same as analytical TLC. The only difference being the compound band along with the silica can be scrapped out, thus purifying them. HPLC is a robust technique for chromatographic separation of analytes at high pressures which improves resolution to a greater extent compared to the conventional techniques (Joseph L. Glajch, 1997 ). In this work, Reverse phase column (C18) and polar solvents were used for identification and detection of the compounds ( Tapan Seal, 2016 )( Klimek-Turek, et al, 2010 ) .
The systems / chemicals used and their corresponding manufacturers are listed below for reference:
· Solvents such as; Hexane and ethyl acetate from RANKEM, Methanol from HI-MEDIA, Acetonitrile and Glacial acetic acid from Merck and MilliQ water from Merck Milllipore apparatus.
· Phenolic acid standards from Sigma-Aldrich.
· Rotary evaporator from Stuart, Cole Parmer.
· TLC Silica gel 60 F254 from Merck.
· Silica gel 60-120 mesh for column chromatography from Sisco research laboratories.
· Silica Gel G for thin layer chromatography from Merck.
· HPLC system from SHIMADZU with C18 column of 5 µm pore size and 4.6 x 250mm dimensions.
A) Extract for isolation of phytochemicals through column chromatography
The leaves of the medicinal plant was collected , oven dried and ground into a fine powder. These were then subjected to Soxhlet extraction. The sample material was packed in thimble and placed in the extraction chamber. The solvents used for extraction were based on increasing polarity namely- Hexane, ethyl acetate and methanol. For each extraction, approximately 250g of sample was loaded as 5 thimbles , which took nearly 75 cycles of Soxhlet extraction with a total solvent volume of 800 ml. Each cycle took place for about 20 minutes approximately depending on the solvent.
B) Extract for HPLC detection
Fine sample powder (1g) was weighed, to which 15 ml of methanol was added. The beaker containing the solution was placed in ice-bath under probe ultrasonication for 40 minutes with 5 seconds pulse on-off cycle at 30% amplitude followed by 3 seconds pulse on-off cycle for 20 minutes at the same amplitude.
The extract obtained using both the methods were subjected to filtration followed by concentration using rotary evaporation. During rotary evaporation, the water bath was set at 65⁰C to provide a constant heat to the sample. The rotor speed was set as 55 rpm , while vacuum was maintained at 800 mbar.
Isolation and purification
Chromatographic techniques were employed for carrying out isolation and purification of plant extract and its compounds.
A) Thin layer chromatography
Thin layer chromatography was used to analyse the components present in a mixture. TLC was done on Silica gel 60 F254, with 20 µl of crude sample loaded on it. The concentration of ethyl acetate in hexane was increased gradually, starting from 5%, 10%, 15%, 20%, 30% till 60%. The spots were detected using iodine staining. These were used as standards for comparing eluted fractions in column chromatography.
B) Column chromatography
The column was packed with silica gel (60-120 mesh) in hexane. 1g of sample was mixed with silica gel and was dry loaded on top of the column. The mobile phase was added onto the column at increasing concentrations of ethyl acetate in hexane. The eluted samples were collected in small volumes in test tubes. The elution of the compounds were detected by performing TLC with the already optimized protocol. After the complete elution, the tubes containing the same compound fractions were pooled together. About 8 fractions were collected and concentrated separately.
C) Preparative thin layer chromatography
For further purification of fractions obtained from column chromatography, preparative TLC was done. The silica gel plates were prepared by pouring a slurry of Silica gel G mixed with deionised water. Alternatively, the silica gel slurry can also be prepared in 10% methanol in ethyl acetate in place of water. The plates are left to dry. Later, the samples were loaded on the plates and run using mobile phase optimized previously. After separation, the compounds are detected by staining in iodine. The desired compounds are then separated from the impurities by scrapping out the silica gel containing that compound. The scrapped out silica is then dissolved in appropriate solvent in which the compounds gets solubilized. The solvent containing the purified compound is then separated from silica gel by filtration.
D) High performance liquid chromatography
High performance liquid chromatography was used to separate, identify, and quantify the components present in extract/ mixture of compounds. In this analysis we performed Reverse phase HPLC for the crude extract sample.
I Detection and separation of structurally similar phenolic acids
For this analysis, four structurally similar compounds were chosen namely- 4- hydroxybenzoic acid (4HBA), Phloretic acid (PA), Phenyl glycolic acid (PGA) and para- coumaric acid (PCA). In order to detect the standards, the samples were scanned over a range of wavelengths using aUV-Vis spectrophotometer. From the data obtained, a suitable wavelength was selected to be 278 nm at which all the standards were adequately detectable at that particular comcentration. Hence, HPLC was performed with the following conditions:
(i) Data acquisition time = 40 mins.
(ii) Detector = 278 nm.
(iii) Column temperature = 25⁰C
(iv) Injection volume = 20 µl
(v) Stationary phase = Silica gel C18 column with 5µm pore size.
(vi) Mobile phase = continuous and stepwise gradient of methanol and MilliQ water with 0.1% Glacial acetic acid.
(vii) Sample concentration = 50µg/ml
(viii) Sample = Mixture of standard phenolic acids.
Initial run was performed with a continuous gradient of the mobile phase at the rate of 2.375% elution per minute. In order to resolve the peaks further, the sample was run with an optimized stepwise gradient at the rate of 0.75% elution per minute. The corresponding standards for the resolved peaks were confirmed by running the standards with the same protocols and the retention times were compared.
II Estimation of para-coumaric acid in the crude extract
For identification of PCA in the plant extract, the retention times of the pure standard PCA was compared with that of the crude extract. The samples were run at 20% acetonitrile in 0.1 % aqueous acetic acid at 280 nm. For quantification of PCA in the same extract, a different protocol was used i.e., 25 % methanol in 0.1 % aqueous acetic acid, for better resolution of PCA peak from other extract compounds. The concentration of PCA was estimated from standard curve obtained by running different concentrations of PCA (5 µg/ml, 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml ) under the same conditions.
RESULTS AND DISCUSSION
(A) Isolation of compounds from leaves of medicinal plant
Identification of compounds by TLC showed nearly 6 bands when stained by iodine.
Column chromatography of the sample yielded 8 fractions containing 6 individual compounds and 2 compound mixtures. Impure fractions were purified using prepartive TLC by running the samples at 40% ethyl acetate in hexane.
(B) HPLC analysis of standard plant phenolic acids
For detection of the standard phenolic acids in HPLC, UV-Vis Spectrophotometric studies of the standard phenolic acids was carried out to identify their corresponding absorbance maxima. The maximum wavelength of absorption for the standards was found to be;
i. PCA = 300 nm
ii. 4HBA = 250 nm
iii. PGA = 250 nm
iv. PA = 224 nm.
A wavelength of 278 nm was chosen for detection of all the phenolic acids. Hence the mobile phase optimisation was carried out at this wavelength.
The retention time of the selected compounds were tested with two differernt mobile phases- 2.375 % methanol in 0.1% acetic acid increase per minute (Protocol 1) and 0.75 % methanol in 0.1% acetic acid increase per minute (Protocol 2). The comparison between Protocol 1 and Protocol 2 for standard phenolic acids is shown below:-
|S.no||Compound||Retention time (Protocol 1) (mins)||Retention time at (Protocol 2)(mins)|
The chromatograms obtained for both the runs are shown below:-
The peaks corresponding to the standard phenolic acids detected using Protocol 1 are illustrated in figure 9.
The retention time of individual standards were found to be around 14 minutes for PGA, 17 minutes for 4HBA, 19 minutes for PA and 23 minutes for PCA. Based on the retention times of the standard and the mixture, the peaks in the mixtures were corresponded to the individual standards.
(C) HPLC analysis of PCA estimation in the plant extract
Plant crude extract was tested for the presence of PCA. The crude sample was run in HPLC at 10µl injection volume, 300nm detector wavelength at 20% acetonirile in 0.1% aqueous glacial acetic acid. The chromatogram obtained is shown in figure 10.
The PCA peak was detected by running a standard 20µg/ml PCA concentration at the same optimized conditions used for running the crude sample. The peak was obtained at a retention time around 13 minutes as shown in figure 11.
For quantification of PCA in the crude extract, 25 % methanol in 0.1 % aqueous acetic acid was used as the mobile phase for better resolution of PCA peak from other extract compounds. The AUC of PCA peak in crude sample under this optimisation was found to be 598634 with retention time around 22 minutes (Data not shown). By substituting this AUC data in the standard equation; the amount of PCA in the crude extract was estimated to be 0.122µg/ml (table 2 and figure 12).
The data obtained from the chromatograms of increasing concentration of standard PCA are tabulated as shown :-
The standard graph was plotted using the AUC data against concentration (figure 10).
Hence the amount of PCA present in the plant extract was estimated successfully.
CONCLUSION AND RECOMMENDATIONS
Extraction by Soxhlet apparatus and ultrasonication was carried out. Filtration removed suspended particulate matter from the extract and rotary evaporation resulted in separation of solvent thereby concentrating the extract. Isolation and purification of compounds by chromatographic methods provided a clear understanding of the number of compounds present in the extract and elution mobile phase concentrations corresponding to each compound. HPLC studies showed the detection of standard phenolic acids and quantification of one such phenolic acid in the crude extract. The optimisation of mobile phases corresponding to the retention time of the peaks were also executed.
Troubleshooting and limitations of the research may include; thermal degradation of extract in Soxhlet apparatus and its solvent losses, long term storage of standards in solution form leads for multiple peaks in the chromatogram, HPLC pressure fluctuations leads to significant differences in the retention times of the compounds and so on.
Further studies may involve estimation of anti-oxidant, anti-microbial, anti-inflammatory and anti-cancerous activity of the purified phytochemicals; followed by their structural characterisation.
First and foremost, I would like to thank The Indian Academy of Sciences to have given me this opportunity to work as a summer intern at the Indian Institute of Technology, Guwahati.
I would like to express my sincere gratitude to my supervisor Dr. Latha Rangan, Professor, Department of Biosciences And Bioengineering, IIT Guwahati, for allowing me to work at the Applied Biodiversity lab by providing humble guidance and independence to work on the title, “Analytical Techniques involved in extraction and purification of phytochemicals from a medicinal plant”. Without her support, nothing would have been possible.
Next, I would love to convey my biggest thanks to my senior S.Sanjana, pursuing Ph.D, Department of Biosciences and Bioengineering, IIT Guwahati; for patiently explaining the handling techniques of analytical instruments, guiding me throughout the complete work and helping me out when encountering a problem, in and out of the lab. I would also like to thank all my dedicated seniors, Anuma singh, Heeramoni Boro, Alok Senapati, Vivian F. Kharchandy, Manish Kumar Gupta and fellow summer intern Padmanav Baruah; for suggesting ideas, having healthy discussions, teaching several methodologies and principles and assisting me throughout the training for my successful timely completion.
I feel happy to render my sincere thanks to the management of Indian Institute of Technology, Guwahati; for providing a clean and comfortable accommodation and healthy food during my course of stay. I also like to acknowledge my room-mate Ankita kumari, and co-interns; Janaani Sri, Dhanya Ramadurai, Madhumitha and Sowmyananjan; for creating a home-like environment at the campus and exchanging opinions on our corresponding research areas. I sincerely thank all of you for your support, care and the courage you have sown in me to explore more places, pursue more trainings and continue my career in Biotechnology with positivity, irrespective of the place I would go.
It would be incomplete if I not thank my home Institution, Sathyabama Institute of Science and Technology; for their strong support in allowing me to attend the summer training. I would love to thank the Management for their encouragement and the freedom they give to the students to attend trainings, learn more and acquire knowledge for our better future. I thank Dr, Swarnalatha, Dean, Higher studies ; who found my capability and insisted me to apply for the summer training; Dr.Ramesh Kumar, Head, Department of Biotechnology; who never fails to support his students and took the responsibilities regarding my attendance; Dr. Reji, Profeesor, Department of Biotechnology, for his guidance and moral support, without which I would have not been a successful intern. I also thank all my teachers, supporting staffs and friends for your well wishes and prayers which greatly influenced me to have a fortunate and successful summer training.
Last but not the least, I feel blessed to thank my parents for kindly allowing me to pursue a summer training far from home. Without your permission, support and strength at the first place, none of this would have been possible.
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