Microwave Supported Extraction and Optimization of Flavonoid Mangiferin from Mangifera indica L. Stem Bark using Orthogonal Array Design

 

Abdul Baseer Khan^1,2, Bhuvaneshwari J¹, Muhammad Arif²*

¹Deptt. of Pharmacognosy, Al Ameen College of Pharmacy, Bangalore - 560027, Karnataka, India.

²Faculty of Pharmacy, Integral University, Kursi Road, Lucknow, 226026, India.

 

ABSTRACT:

A modest and swift microwave-backed extraction process has been established for the abstraction of a flavonoid mangiferin from the Shoot bark of Mangifera indica L. The content in the extracts was quantified by planar chromatography. An orthogonal array strategy was employed to conclude the effects of variables that affect the efficiency of MAE, namely temperature, irradiation period, the clout of irradiation, and particle size of drug powder. Under optimal conditions, MAE showed expressively higher recovery of mangiferin and markdown in the extraction period as well as solvent intake in comparison to the conventional method.

 

 

 

INTRODUCTION: 

Mangifera indica L. is a bulky perennial tree whose bark is extensively used in traditional medicine¹.  The bark is specified to encompass mangiferin, protocatechic acid, catechin, glycine, alanine, γ-aminobutyric acid, shikimic acid, kinic acid, along with the tetracyclic triterpenoids cycloart-24-en-3β, 26diol, 3-ketodammar-24 (E)-en-20S,26-diol, C-24 epimers of cycloart-25 en 3β,24, 27-triol and cycloartan-3β,24,27-triol². Mangiferin is a widely distributed, stable flavanol c-pyranoside with 1, 3, 6, 7-tetrahydroxy xanthone as the aglycone³. It is reported for antidiabetic, anti-inflammatory, anti-tumor, immune-modulator, anti-hyperlipidemic, anti-atherogenic, anti-bone resorption, monoamine oxidase-inhibiting, and antiviral properties^4-7. The compound has been isolated by different methods from different sources.  One of the extensively employed techniques for the isolation of mangiferin involves the abstraction and refinement of mangiferin from the dried stem bark of Mangifera indica using a series of solvents⁸.

 

 

Another method makes use of pH-controlled extraction by partitioning between different solvents followed by purification using a cephadex column from mango pure concentrate⁹. Leaves of M. indica have been utilized for the abstraction of mangiferin by refluxation of defatted material using ethanol and crystallization from ethanolic extract using ethyl acetate¹⁰. Various conditions affect the extraction process, yield, and purity of the compound. Besides, most conventional methods of extraction pose the problem of solvent consumption in large quantities. This makes the process uneconomical and may precipitate toxicity. Such methods are also time-consuming, run a risk of degradation of phytoconstituents during extraction. There has been an evolution of newer methods of extraction with reduced extraction time, compact solvent consumption, and pollution anticipation concerns. One such method is the use of microwave-assisted extraction (MAE) which heralds the practice of green pharmacy¹¹. The use of microwave energy-assisted heating for the effective extraction of phytoconstituents is now gaining popularity. MAE of mangiferin from leaves of Mangifera indica is reported¹². The main purpose of MAE is a reduction of time and solvent quantity.  The high temperature of microwave can hydrolyze the ether linkages of the cellulose matrix and helps the solvents access the compound inside the cell easily.  Various factors such as the nature of the solvent, its volume, extraction time, microwave power, irradiation time, particle dimension of the plant material, and temperature affect the abstraction process. In the present study, we have studied the microwave extraction efficiency for mangiferin commencing from the shoot bark of Mangifera indica and the content of mangiferin in extracts quantified by the planar chromatography method, and an orthogonal array design procedure was used to conclude the effects of variables that alter the efficiency of MAE.

 

MATERIALS AND METHODS:

Assortment and authentication:

The shoot bark of Mangifera indica L. was gathered from dedicated groves in Madanapalli, Andhra Pradesh, and authenticated by Dr. Shiddamalaya Raman Research Institute, Bangalore. A voucher variety No-85472 was reserved in the research laboratory for supplementary allusion.

 

Chemicals:

Mangiferin 98% pure was procured from Sigma Aldrich, Laboratory grade solvents were used for extraction, and HPLC grade solvents from SD fine chemicals were used for chromatographic determination. Silica gel 60 F₂₅₄ TLC plates from Merck were for HPTLC.

 

Conventional extraction method:

The collected bark was air-dried and ground using a mixer grinder and powders of mesh size 16 were used for preliminary extraction. A three-step solvent extraction method was optimized for extraction of mangiferin 50g of powdered bark was subjected to de-fating by refluxation using petroleum ether for eight hours and detannified using cold acetone for six hours three times and finally extracted with 70% ethanol (aqueous) by macerating for 24 hours two times and the extract was collected and every time before extraction with subsequent solvent the powdered material was air-dried¹³.  A solid to solvent ratio of 1:10 was maintained. The extract was concentrated by using a Rotary Vaccum evaporator at a low temperature. It was then air-dried and the yield calculated.  For any set of extraction conditions, extractions were repeated three times and an average of three yields is reported in table 1.

 

Optimization of extraction for mangiferin:

For obtaining mangiferin enriched extract from M. indica bark powder, we have studied the effects of particle size of drug powder, microwave power, and irradiation time on the extractability of mangiferin with 70% ethanol. An orthogonal array design similar to the method adopted by Sevil Banay Razi et al has been employed¹⁴. The scheme followed for MAE from M. indica is reported in table 2.

 

Preparation of extract by MAE:

For MAE accurately weighed quantity of 10g each of powders of sieve number 12, 22, or 44 of Mangifera indica was taken in the borosilicate round bottom flask along with 70% aqueous ethanol (50ml). The sample was retained in the microwave cavity and irradiated with microwave for the diverse time of exposure and at altered power levels as outlined in table 2. To avoid charring of the sample it was stirred continuously at a constant speed for all above trials till completion of the process. After extraction drug was filtered through Whattman filter paper Number 1, the filtrate was air-dried and the yield of extractive was determined.

 

Determination of mangiferin content of extracts by planar chromatography:

HTLC procedure was developed using different solvent systems as mobile phase. The Solvent system that gave good resolution, sharp and well-defined peak was selected. The spotted plate was developed in a twin trough chamber at room temp. The plate was scanned in the range of 200-600nm using D2 and a tungsten lamp to determine λ_max. The method was optimized concerning plate saturation time and length of development. The linearity range for mangiferin was determined using Standard Mangiferin 5mg dissolved in 1ml of DMF and diluted to10ml with methanol to give a concentration of 0.5mg/ml.  2μl -12μl of the solution was applied using Linomat 5 as bands.  The plate was developed to the length of 80mm and scanned at 260nm. The peak area/ height data obtained was checked for linearity in linear mode. The Limits of detection and quantification were determined^15-16.

 

Table 1: Yield of extract and its mangiferin content from Mangifera indica  by conventional extraction method

 

RESULTS AND DISCUSSION:

The average yield of extracts and their mangiferin content by the conventional method are shown in table 1. We have studied the effects of particle size of drug powder, microwave power, and irradiation time on the extractability of mangiferin with 70% ethanol by MAE. An orthogonal array design was adopted for the study.  Results are recorded in table 2.

 

 

Table 2:  Yield of extract and mangiferin content from M. indica by Microwave-assisted extraction with orthogonal test L9 (3³) design

* 5μl of 1μg/μl solution of mangiferin enriched extract obtained under different conditions as outlined in the table

 

A consequence of microwave power:

The impact of microwave power on the yield of extract stood at power levels of 40%, 60%, and 80%; it was observed that at the 40% power level there was not much change in the yield of the extract by varying the time of extraction and particle size. Whereas, altering these parameters at power levels of 60% and 80% affected the yield of the extracts. A direct linear correlation was observed between the yield of extract and increased power levels keeping the particle size constant. 

 

Effect of irradiation time:

The yield of extract increased when extraction time was increased from 1 to 2 minutes.  The maximum amount of extract was obtained with an irradiation time of 2min under the conditions tested, and the yield of extract dropped slightly when irradiation time was increased to 4min. This indicates that an irradiation time of 2min is sufficient to maximize the extraction of mangiferin and the reduction in extractive content may be due to the decomposition of the constituent at elevated temperature for an extended period.

 

Effect of particle magnitude:

The particle size of the drug matrix and the conditions under which it is irradiated with microwaves affect the effective recoveries of the biopharmaceuticals. Considering the effect of particle size on the yield of extract, it was seen that with powder #12, the yield of extract was inversely affected by power level.  However, with powder #22, the yield of extract was directly related to power level and with powder #44, the yield of extract increased up to power level 60%; but reduced at power level 80%. (Figure-1)

 

Fine powder can augment the extraction by ensuring extended surface area, which further enhances contact among the plant matrix and the solvent, also smaller particles improve the subterranean penetration of the microwave and one of the shortcomings related to finer particles is a skirmish of the parting of the matrix from the solvent subsequently to the microwave irradiation¹⁷. With the use of fine particles, deep penetration of microwaves will be facilitated resulting in thermal degradation of active constituents¹⁸. This may explain the reduced yield with powder of #12. 

 

 

Figure.1. The yield of mangiferin in 100 g extract, and bark powder of different particle sizes after MAE using the L9 design

 

According to the largest donating rule, to determine the effects of each investigated factor, the conditions yielding the largest content of extract/mangiferin should be the selected value to determine process optimization. The largest value was taken as the optimized value. Therefore, the optimized experimental conditions for MAE were the microwave power of 80%, irradiation time of 2min, and powder of mesh size-22. Under the optimum conditions, the total extraction yield was 39.9%. However, quantification by planar chromatography, revealed that conditions of the 7^th group, namely microwave power- 40%, irradiation time- 1min, and powder of mesh size- 44 was more selective for mangiferin since mangiferin content of the extract was higher than with conditions of 2^nd group (67.04%as against 53.08%). Ultimately considering both yields of extract and its mangiferin content, conditions of 2^nd group extract the maximum amount of mangiferin from a fixed mass of M. indica powder matrix.  This indicates that the extraction yield of mangiferin can be enhanced using a combination of these factors at different levels in the extraction process.

 

Figure 2. 3D diagram of HPTLC densitogram of mangiferin in M. indica extract obtained from 9 levels of MAE.

 

 

To develop an HPTLC method for the determination of mangiferin from the extracts, we attempted using different mobile phases. The best mobile phase for development was found to be Ethyl acetate: acetic acid: Formic Acid: Water (100:11:11:26). R_F with the above mobile phase was 0.57.  λ max was 260nm.  The LOD for mangiferin by the HPTLC method developed was found to be 32.5 ng (peak area = 192.6); The LOQ was therefore 97.5 ng (peak area = 577.8). Linearity was good between0.075 μg to 12 μg. (Figure-2).

 

Factors affecting the yield of mangiferin by conventional and MAE methods on M. indica:

Optimization strategy for both conventional and MAE was attempted. Optimization for the conventional method was more time-consuming, utilized more solvents and power. A comparative account concerning mangiferin content in extracts, % yield of mangiferin enriched extracts and mangiferin extractable from 100 g of drug from conventional and MAE methods is summarized in table 3.

 

MAE activates cell teeming due to confined inner superheating tracked by leaching out of the dynamic ingredients¹⁹. Cell teeming accelerate the entrance of the mining solvent to solubilize the ingredients, thus priming the quicker and more efficient extraction. When matched with orthodox extraction methods, MAE also is superior concerning control of temperature; power level, effective distribution of heat (aided by convection) in the oven as well as stirring.  It is also environment-friendly and cost-effective. Analogous reports related to a decline in extraction time and solvent volume has been testified for MAE of several natural products²⁰.

 

Table 3: Comparison between conventional and Microwave-assisted extraction methods

* = Optimized conditions

 

The mangiferin was isolated by recrystallization using 70% ethanol as a yellow amorphous powder²¹. The isolated compound was characterized by UV, IR, HNMR and LC/MS, and TLC. The findings of the above-mentioned analysis are very closely matched with that reported for standard mangiferin. Hence it was concluded that the isolated biopharmaceutical compound was mangiferin.

 

CONCLUSION:

MAE technique for sequestration of Mangiferin was augmented using orthogonal array design and aid in the identification of finest values for fickles that can significantly alter the extraction with a restricted number of experimentations. The quantity of mangiferin extracted is vastly dependent on microwave power, irradiation time along with particle size of the material used for extraction. Compared with the orthodox extraction technique employed in this learning, the proposed MAE procedure was superior concerning the economy of time solvent and power utilization. Thus attempts to scale up MAE for commercial extraction of biopharmaceuticals; application of this method in sample preparation for analysis of marker compound/impurity and extension of similar methods for stability analysis of herbal extracts and formulations may reveal more efficient methods of biopharmaceuticals extraction and analysis.

 

ACKNOWLEDGEMENTS:

The authors wish to thank Al-Ameen college of Pharmacy, Bangalore for providing the required facilities to carry out the work.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

REFERENCES:

1.     Wauthoz N., Balde A., Balde ES., Van Damme M., Duez P. Ethnopharmacology of Mangifera indica L. bark and pharmacological studies of its main C-glucosylxanthone, mangiferin. International Journal of Biomedical and Pharmaceutical Sciences, 2007, 1(2), 112-119, Corpus ID: 54713735.

2.     Scartezzini P., Speroni E. Review on some plants of Indian traditional medicine with antioxidant activity. Journal of Ethnopharmacology 2000, 1, 23-43, 10.1016/s0378-8741(00)00213-0.

3.     Muruganandan S., Gupta S., Kataria M., Lal J. Gupta PK. Mangiferin protects the streptozotocin-induced oxidative damage to cardiac and renal tissues in rats. Toxicology 2002, 176, 165-173, 10.1016/s0300-483x(02)00069-0.

4.     Sanchez GM. Re L. Giuliani A.Nuñez-Selles AJ. Davison GP. Leon-Fernandez OS. Protective effects of Mangifera indica L. extract, mangiferin and selected antioxydants against TPA-induced biomolecules oxidation and peritoneal macrophage activation in mice. Pharmacological Research, 2000,42, 565-573, 10.1006/phrs.2000.0727.

5.     Arivukkarasu R. Rajasekaran A. Hussain SHB., Ajnas M. Simultaneous detection of Rutin, Quercetin, Gallic acid, Caffeic acid, Ferulic acid, Coumarin, Mangiferin and Catechin in Hepatoprotective commercial herbal formulations by HPTLC technique. Research Journal of Pharmacognosy and Phytochemistry, 2018 10(1), 59-62.

6.     Yoshikawa M, Shimoda H, Nishida N, Takada M, Matsuda H. Salacia reticulata and its polyphenolic constituents with lipase inhibitory and lipolytic activities have mild antiobesity effects in rats. The Journal of Nutrition, 2002, 132, 1819-1824, 10.1093/jn/132.7.1819.

7.     Zhu XM. Song JX. Huang ZZ. Whu YM. Yu MJ. Antiviral activity of mangiferin against herpes simplex virus type 2 in vitro. ZhongguoYaoli Xuebao,1993,14, 452-454.

8.     Arivukkarasu R., Rajasekaran A. Detection and Quantification of Anti-oxidant markers like Rutin, Catechin, Quercetin, Gallic acid, Ellagic acid, Ferulic acid, Vitexin and Mangiferin in Herbal raw materials available in market belongs to Rutaceae family by HPTLC Technique. Asian Journal of Pharmaceutical Analysis, 2021,11(2) 151-155, 10.52711/2231-5675.2021.00026.  

9.     Schieber A. Ullrich W. Carle R. Characterization of polyphenols in mango puree concentrate by HPLC with diode array and mass spectrometric detection. Innovative food science and emerging technologies 2000;1:161-166, 10.1016/S1466-8564(00)00015-1.

10.  Upadhye M. Gandhi P. Phanse M. Pharmacognostical, Phytochemical Studies and Comparative Extraction Techniques of Daucus carota. Research Journal of Pharmacognosy and Phytochemistry, 2014, 6(3), 115.

11.  Arif M. Fareed S and Hussain MS. Estimation of antioxidant activity of microwave assisted extraction of total phenolics and flavonoids contents of the fruit Spondias mangifera. Asian Journal of Traditional Medicines, 2011, 6 (4) 146-155.

12.  Krishnan C. Mullapudi V B K. & Garg N. Development of a single-step microwave-assisted digestion method using dilute nitric acid for determination of bismuth in bismuth-containing pharmaceuticals by hydride generation-atomic fluorescence spectrometry. Asian Journal of Pharmaceutical Analysis, 2021, 11(2)87-7, 10.52711/2231-5675.2021.00017.

13.  Arif M. Zaman K and Fareed S. Pharmacognostical studies and evaluation of total phenolic contents of trunk bark of Spondias mangifera Willd. Natural Product Radiance, 2009; 8(2):146-150. 10.2298/ABS1102413S

14.  Razi S B. Zaaeri F. &Javar H A. An HPLC Method for detection of Anti-inflammatory Drugs in Bone and Cartilage health supplements. Asian Journal of Pharmaceutical Analysis, 2020, 10(2), 67-76.

15.  Singh P. Arif M. Qadir A. &Kannojia P. Simultaneous analytical efficiency evaluation using an HPTLC method for the analysis of syringic acid and vanillic acid and their anti-oxidant capacity from methanol extract of Ricinus communis L. and Euphorbia hirta L. Journal of AOAC International, 2021, 104(4), 1188-1195, 10.1093/jaoacint/qsaa171.

16.  Karpakavalli M. Badami S. Allimalarkodi S., Christi V E. Microwave Assisted Isolation of Dye from Heartwood of Artocarpous heterophyllus Lam. Research Journal of Pharmacognosy and Phytochemistry, 2010, 2(4), 303-305.

17.  Kwon JH., Lee GD., Bélanger JM., Jocelyn Paré JR. Effect of ethanol concentration on the efficiency of extraction of ginseng saponins when using a microwave‐assisted process (MAP™). International journal of food science & technology, 2003, 38(5), 615-622.

18.  Selvam TP., Saravanan G., Prakash CR., Kumar PD. Microwave-Assisted synthesis, characterization and biological activity of novel pyrazole derivatives. Asian Journal of Pharmaceutical Research, 2011, 1(4), 126-129.

19.  Ehsan S., Khan B. Conventional and microwave-assisted synthesis of 5-halogeno-(X) 2, 4-(1H, 3H) pyrimidinedione and their biological evaluation. Asian Journal of Research in Chemistry, 2010, 3(4), 1103-1106.

20.  Dinesh D., Rishipathak Trupti A., Jadhav Sonal P., Tathe Pavan B. Udavant. Microwave Assisted Synthesis and Pharmacological Evaluation of Few 4-Quinazolinone

 

 

 

Accepted on 18.06.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1113-1117.