Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Chemical Constituents and Biological Functions of Different Extracts of Millettia speciosa Leaves

N Nasiruddin, Minghui Ji, Zhangxin Yu, Guangying Chen*, Tariq Masood, Funing Ma

Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal University, Haikou 571127, P. R. China

Abstract: Millettia speciosa, a traditional Chinese folk medicine, used as immunity enhancer and anti-bronchitis. The study was proposed to investigate phytocomponents and pharmacological activities (antityrosinase, sunscreen and anticancer) of the different fractions of M. speciosa leaves. The sample was extracted with solvents of increasing polarity i.e. petroleum ether (PE), ethyl acetate (EtOAc), methanol (MeOH) and the total flavonoid content for each fraction was determined. The GC-MS analysis revealed various important bioactive compounds. The identified major compounds included beta sitosterol (12.039%), (Z)-9-octadecenamide (6.299%) and gamma sitosterol (4.910%). The highest flavonoid content was recorded for the PE extract i.e. 47.50 ± 0.40%. The PE extract showed significant antityrosinase activity (70.30 ± 0.90 %) with IC50 0.035 mg/ml followed by the MeOH extract i.e. 66.50 ± 1.20% with IC₅₀ 0.039 mg/ml. The highest sunscreen activity was noted for the EtOAc extract by observing maximum absorbance value (1.518 ± 0.49) in UVA region followed by 1.214 ± 0.46 in UVB zone. The highest cytotoxicity (29.45 ±1.17% viability) towards MCF-7 breast cancer cells line was noted for the PE extract with IC50 6.73 µg/ml. Overall, our study revealed the presence of potential bioactive compounds for each extract that exhibited significant therapeutic activities and therefore validate the importance of M. speciosa leaves as a source of natural medicine.

Keywords: Millettia speciosa, GC-MS analysis, bioactivities, flavonoid, sunscreen, antityrosinase, anticancer

Received August 12, 2020; Revised September 15, 2020; Accepted September 24, 2020

Citation: N Nasiruddin, Minghui Ji, Zhangxin Yu, Guangying Chen, Tariq Masood, Funing Ma, Chemical Constituents and Biological Functions of Different Extracts of Millettia speciosa Leaves. Journal of Food and Nutrition Research, 8 (9). 506-515. January 2020. doi:10.12691/jfnr-8-9-7

1. Introduction

Medicinal plants serve as an important source of new drug leads due to their diverse phytochemical composition. Many studies have confirmed that medicinal plants contain a number of biologically important compounds like phenols, alkaloids and other compounds soluble in water and organic solvents [1]. While, other works have characterized the medicinal values of plants which may include anticancer, antidiabetic and antioxidant properties, etc. Different extracts of different plants, can show a wide range of medicinal effects such as hepatoprotection, antimalarial activity, antibacterial and many others [2]. About 80% people living in developing world use herbal medicines as a primary and traditional healthcare source. During the past decade, a remarkable public acceptance and interest for natural therapies has been witnessed both in developing and developed countries [3]. Extracts obtained from various parts (stems, leaves, roots, flowers etc.) are widely used for in vitro and in vivo scientific studies. To date, approximately 500, 000 plant species have been reported around the globe, and only 1% has been investigated for phytochemicals. This situation creates a huge potential to search for unexploited plants and discover new therapeutic compounds [4].

M. speciosa is a tropical plant of Leguminosae family, which is widely distributed in China, tropical and sub-tropical regions of the world. The plant has been reported to be used as immunity enhancer and anti-bronchitis [5]. However, no detail research studies on GC-MS analysis and pharmacological activities of M. speciosa leaves have been reported so far. In present study, we report phytochemical composition, tyrosinase inhibition, sunscreen and anticancer activities of petroleum ether (PE), ethyl acetate (EtOAc) and methanolic (MeOH) extracts of M. speciosa leaves.

2. Material and Methods

2.1. Collection of Plant Material

Millettia speciosa was collected from Wanning city, Hainan province in May 2018, and was identified by Professor Zhong Qiongxin, College of Life Sciences of Hainan Normal University, Haikou, China. The sample was stored in Hainan Key Laboratory of Tropical Medicinal Plant Chemistry, College of Chemistry and Chemical Engineering, Hainan Normal University having sample number (20110511MS).

The leaves of M. speciosa were selected for the study. The collected sample was washed with tap water to remove dust. The sample was dried in shade for several days till complete dryness. The dried sample was grounded into powder with lab-crusher HS-400Y and kept in an air tight container for future use.

2.2. Extraction

Extraction of dried powder was carried out by maceration using different organic solvents of increasing polarity (petroleum ether, ethyl acetate and methanol). A 300 gm of each sample was separately and sequentially extracted. The samples were immersed in 5 L petroleum ether for 3 days and filtered through fresh cotton plug and finally with Whatman filter paper. The filtrate was concentrated using rotary evaporator. The residue from first step was immersed back in 5 L ethyl acetate for 3 days. Same procedure was followed to get raw extract of ethyl acetate. The residue from second immersion was extracted with methanol, resulted in raw methanolic extract. The raw extracts were kept in an air tight container at 4°C till further study [6].

2.3. GC-MS analysis

The procedure for GC-MS analysis of crude extracts of M. speciosa leaves was followed as described by Mustapha et al. [7]. Briefly, analysis were carried out on equipment model Agilent 7890A/5975C using capillary column operating in EI mode. An HP-5 MS column (30 m x 250 μm x 0.25 μm) was used. Helium was used as a carrier gas at a constant flow rate of 1.2ml/min. The percent composition of chemical constituents in crude extracts was expressed as percentage by peak area. The identification and characterization of compounds was based on their retention indices. Mass spectra were obtained by EI at 70 eV over the scan range 35-650amu. The mass spectra were matched with standards available in NIST 08.L Mass Spectrum libraries.

2.4. Determination of Total Flavonoids

The total flavonoids content was determined by using spectrometry method [1,8] with slight modifications. Rutin was used as Standard to prepare stock solution. A series of different dilution was made to establish a linear equation based on a calibration curve. y=10.818x-0.0217, R²=0.9975. Where “y” is the absorbance and ‘’x’’ is the flavonoids content in mg/ml.

The extracts were dissolved with dimethyl sulfoxide. In a 10 ml test tube, 2ml of extracts, 3.4 ml of 30% methanol, 0.15 ml of NaNO₂ (0.5 M) and 0.15 ml of AlCl₃.6H₂O (0.3 M) was mixed. After 5 min, 1 ml of NaOH (1 M) was added. The solution was mixed well and the absorbance was measured against the reagent blank at 510 nm. Samples were run in triplicate and mean values were calculated as mg rutin per g dry weight (% w/w). The total content of flavonoid was calculated using the following formula;

Where, RE is the concentration of rutin solution obtained from the calibration curve; V, is the volume of extract (ml) and m, is the weight of pure leaf extract (g).

2.5. Antityrosinase Activity

The tyrosinase inhibitory activity of all extracts was determined using previously described method [9] with some modifications. In short, 1 ml of potato tyrosinase was added to phosphate buffer (pH= 6.8) and 2 ml of L-tyrosinase and 2 ml of different concentration of leaf extracts. The experimental reaction time was 30 min at 37°C. After incubation, absorbance of the mixture was measured at 490 nm using UV-spectrophotometer. Arbutin (0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 mg/ml) and Vitamin C (0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 and 0.4 mg/ml) was used as positive control. The percent inhibition of tyrosinase was calculated using the following formula;

2.6. Sunscreen Activity

The spectrophotometric method was used to determine sunscreen activity as described earlier but with some modifications [10,11]. The test solutions were prepared by taking 100-200 μg/ml concentration. The absorbance was measured in UVC (200-280nm), UVB (280-320nm) and UVA (320-400nm) region using dual beam ultraviolet-visible spectrophotometer (TU-1901, Beijing General Analysis Instruments). Rutin and 4-methylbenzylidene camphor (sunscreen) was used as positive control.

2.7. Anticancer Activity

2.7.1. Cell Culture

MCF-7 cells (human breast cancer cell line) were procured from Kunming Cell Bank, Chinese Academy of Sciences, China. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics penicillin (5000 units/mL), which were purchased from Gibco, Invitrogen, CA, USA. Cells were maintained at 37°C in 5% CO₂ humidified incubator. Cells were subcultured at 80% confluency, and fresh media was applied every 2-3 days interval [12,13,14].

2.7.2. Evaluation of Cellular Cytotoxicity

The cytotoxicity was evaluated by the crystal violet staining (CVS) method with some modification [15,16]. In short, the cells were seeded in a 96-well tissue culture microplate, at a cell concentration of 4 × 10³ cells per well in 100 μl of growth medium. Fresh medium containing different concentrations of each fraction (i.e. PE, EtOAc and MeOH) was added after 24 hrs of seeding at 37°C. Serial two-fold dilutions (200 μg/ml, 100 μg/ml, 50 μg/ml, 25 μg/ml and 12.50 μg/ml) of the extracts were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates using a multichannel pipette. The microtiter plates were incubated at 37°C in a humidified incubator with 5% CO₂ for a period of 72 hrs. Three wells were used for each concentration of the test sample i.e. the samples were tested in triplication. Control cells (MCF-7 cells with medium) were also incubated without test samples.

At the end of the incubation period, media was aspirated and the crystal violet solution (100 μl of 1%) was added along with fetal bovine serum (1:4.5) to each well for at least one and half h. The stain was removed and the plates were rinsed using distilled water. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly. The viable cells yield was determined by a colorimetric method. The percentage of viable cells was calculated by using formula; % Viability= absorbance of treated cells/absorbance of control cells *100 [13]. The quantitative analysis (colorimetric evaluation of fixed cells) was performed by absorbance measurements in an automatic Microplate reader (Thermo Scientific Multiskan Go) at 590 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. The effect on cells growth was calculated as the difference in absorbance percentage in presence and absence of the test extracts. The results were illustrated in a dose-response manner. The IC₅₀ (concentration at which 50% growth inhibition of cancer cells occurs) was calculated. Doxorubicin was used as the standard anticancer drug.

3. Results

3.1. GC-MS Analysis

GC-MS chromatograms were obtained for extracts of solvents with increasing polarity (petroleum ether, ethyl acetate and methanol) are given in Figure 1 - Figure 3, respectively. GC-MS chromatogram of the PE extract depicted different peaks resulting the presence of 54 compounds (Table 1). The most abundant compounds identified were gamma sitosterol (4.910%), followed by n-hexadecanoic acid (4.745%), stigmast-4-en-3-one (2.939%) and gamma tocopherol (2.845%).

The peaks in the chromatogram of the EtOAc extract resulted in 14 compounds identified according to their retention time (Rt) on capillary column (Table 2). Out of these compounds, beta sitosterol (12.039%) was found a major chemical constituent. This was followed by (Z)-13 docosenamide (4.270%), campesterol (2.705%), 2-pentadecanone, 6, 10, 14-trimethyl (2.046%) and phytol (1.787%).

The GC-MS analysis of the MeOH extract of M. speciosa leaves led to the identification of 12 chemical compounds (Table 3) with higher similarity index. Among these compounds, beta sitosterol (9.860%) was characterized and identified as the most significant phytochemical. Other major compounds observed were (Z)-9-octadecenamide (6.299%), stigmasterol (6.071%) and n-hexadecanoic acid (4.316%).

Table 1. GC-MS analysis of petroleum ether extract of Millettia speciosa leaves

Table 2. GC-MS analysis of ethyl acetate extract of Millettia speciosa leaves

Table 3. GC-MS analysis of methanolic extract of Millettia speciosa leaves

Figure 1. Total ion chromatogram for GC-MS analysis of petroleum ether extract of leaves

Figure 2. Total ion chromatogram for GC-MS analysis of ethyl acetate extract of leaves

Figure 3. Total ion chromatogram for GC-MS analysis of methanol extract of leaves

3.2. Total Flavonoids

The results for total flavonoids of different extracts of M. speciosa leaves are given in Table 4. The highest amount (P < 0.05) of flavonoids were recorded for the PE extract (47.50 ±0.40%), followed by the MeOH extract i.e. 45.00 ±0.30%. Whereas, the least amount (26.70 ±0.30%) was calculated for the EtOAc extract.

Table 4. Total flavonoid content of various Millettia speciosa leaves extracts

3.3. Antityrosinase Activity

As tyrosinase is an important enzyme for melanin synthesis, the plant material which has inhibitory activity for tyrosinase is thought to be a best candidate for skin whitening and protection against sun-related injuries. Hence, we were interested to investigate the antityrosinase activity of different M. speciosa leaves extracts, arbutin and vitamin C at various concentrations (Table 5, Figure 4). The results indicated highest tyrosinase inhibitory activity (70.30 ± 0.90%) for the PE extract at 0.090 mg/ml concentration with IC50 value 0.035 mg/ml. This was followed by higher value (66.50 ± 1.20 %) noted for the MeOH extract at 0.090 mg/ml concentration with IC50 value 0.039 mg/ml. While, the lowest inhibition (33.33 ± 0.86%) was calculated for the MeOH extract at 0.015 mg/ml concentration.

3.4. Sunscreen Activity

The absorbance values for sunscreen effect of M. speciosa leaves were measured and given in Table 6 and Figure 5. The EtOAc extract exhibited maximum absorbance (1.518 ± 0.49) in UVA region (320~400 nm) at concentration of 200 μg/ml as compared to rutin (1.014 ± 0.28) and 4-methylbenzylidene camphor (0.512 ± 0.19) at 40 μg/ml concentration. Similarly, maximum absorbance (1.214 ± 0.46) in UVB region was also observed for the EtOAc extract but less than 4-methylbenzylidene camphor (3.350 ± 0.52) at given concentrations. While in UVC region, maximum absorbance (0.863 ± 0.38) was recorded for the PE extract but less than rutin (1.908 ± 0.61) and 4-methylbenzylidene camphor (3.485 ± 0.46). The least absorbance (0.459 ± 0.10) was measured for MeOH extract at 320~400 nm.

Table 5. Antityrosinase activity and IC50 (mg/ml) of various Millettia speciosa leaves extracts

Figure 4. Antityrosinase activity of Millettia speciosa leaves extracts, Arbutin and Vit C. Values are expressed as means ± SD (n=3)

Table 6. Sunscreen activity of various extracts of Millettia speciosa leaves

Figure 5. Sunscreen activity of Millettia speciosa leaves extracts, rutin and camphor. Values are expressed as means ± SD (n=3)

3.7. Anticancer Activity

During the last decade, medicinal plants with significant anticancer potential has immensely attracted scientist to develop new therapeutics. In present study, the anticancer effect of different extracts of M. speciosa leaves against MCF-7 cells was investigated. A dose dependent effect was noted as given in Table 7. The experiment was run in triplicate for different concentrations ranged from 12.50-200 μg/ml.

Table 7. Cells Viability (%) and IC50 (μg/ml) for various Millettia speciosa leaves extracts

Figure 6. Anticancer activity of Millettia speciosa leaves extracts and Doxorubicin. Values are expressed as means ± SD (n=3).

The most significant effect (29.45 ± 1.17%) was shown by the PE extract at 200 μg/ml with an IC₅₀ value of 6.73 μg/ml. This was followed by the EtOAc extract (43.51 ± 1.25%) at the same concentration level with an IC₅₀ value of 4.14 μg/ml. While, at highest concentration, the cell viability was reduced to 53.44 ± 1.11% which revealed the least effect of the MeOH extract at IC₅₀ 2.55 μg/ml. The % viability of MCF-7 cells at different strength of M. speciosa leaves extract is given in Figure 6.

4. Discussion

4.1. GC-MS Analysis

To know the medicinal importance of any plant, the initial study is to investigate phytochemical constituents. It gives broad information about nature of phytoconstituents belonging to various classes to develop health promoting novel drugs. The presence of different classes of bioactive compounds e.g. alkaloids, steroids, phenolics, steroids etc. in plant extract have been reported to demonstrate important therapeutic activities [17].

For the first time, various extracts of the leaves of M. speciosa were investigated for bioactive compounds. In the present study, GC-MS analysis of the PE extract revealed 54 compounds. According to the results, gamma sitosterol was found with maximum relative content value. The compound has an important role to induce G2/M cell cycle arrest and apoptosis in MCF-7 and A549 cells [18]. Other major compounds included; n-hexadecanoic acid, stigmast-4-en-3-one and gamma tocopherol. n-hexadecanoic acid has been found responsible for anticancer cytotoxic potential [19]. Stigmast-4-en-3-one is a sterol that exhibits antitumor, antidiabetic and hypoglycemic activities [20]. While anti-inflammatory and antioxidant properties have been reported for gamma tocopherol [21].

Likewise, GC-MS analysis of the EtOAc extract predicted beta sitosterol being a main component. The compound has various pharmacological activities like; anti-oxidant, anticancer, immunomodulatory etc. [22]. While, (Z)-13-docosenamide and phytol are reported to have antinociceptive and anti-inflammatory properties [23]. Similarly, campesterol is responsible for anticancer, antioxidant, cholesterol lowering and antidepressant activities. The compound 2-Pentadecanone, 6,10,14-trimethyl- has anti-inflammatory, cardio-protective, antioxidant and antibacterial capabilities [24].

The compound beta sitosterol and n-hexadecanoic acid was also noted for the MeOH extract. The reports suggested strong antioxidant and antimicrobial properties for (Z)-9-Octadecenamide [25]. While stigmasterol was reported to have anticancer, anti - inflammatory, antioxidant, diuretic and antimicrobial properties [26]. This is the first study to investigate various extracts of Millettia speciosa leaves for bioactive compounds through GC-MS analysis. The presence of various therapeutic compounds in M. speciosa leaves with significant biological activities suggests its pharmaceutical importance and further detailed study for new drug development.

4.2. Total Flavonoid Content

Flavonoids are plant secondary metabolites that exhibit different biological activities due to free hydroxyl groups [27]. The total flavonoid content of different extracts of M. speciosa leaves was colorimetrically determined. Each extract showed different amount of flavonoid content. Our experiment recorded highest amount for the PE extract, while the least for EtOAc extract. The flavonoid content of M. speciosa leaves extracted by PE, EtOAc and MeOH cannot be compared with other results due to unavailability of suitable literature about selected plant and part. However, several reports are available in literature about other medicinal plants. Previously, Sushant et al. revealed 37.86 ± 0.53 mg QE/g in the MeOH extract of Cassia tora [27]. Similarly, 23.25 ± 0.11 μg Qu/g, 4.04 ± 0.23 μg Qu/g and 20.44 ± 0.26 μg Qu/g was recorded for aerial parts of Hyoscyamus gallagheri extracted with hexane, EtOAc and MeOH, respectively [28]. It is clear from the literature that the total flavonoid content of medicinal plants is significantly affected by genetic, biological and environmental variations [29].

4.3. Antityrosinase Activity

Tyrosinase is a copper containing glycoprotein, which plays an important role in melanogenesis. The enzyme catalyzes hydroxylation of monophenols to diphenols and oxidation of o-diphenols to o-quinones to finally form melanin resulting various skin disorders like melasma, age spots etc. [30,31]. Thus tyrosinase inhibitors from natural source are considered attractive targets in cosmetics. In our study, different extracts of M. speciosa leaves were investigated for tyrosinase inhibition. The tyrosinase inhibition was noted in a dose-dependent manner and highest inhibition (70.30 ± 0.90%) was recorded for the PE extract at 0.090 mg/ml concentration, followed by MeOH extract (66.50 ± 1.20) at the same dose. The inhibition could be due to cooper chelating agents present in plant extracts, which prevents cooper ions to bind with oxygen, thereby leading to deactivation of tyrosinase enzyme [32]. It is also assumed that tyrosinase inhibition may be due to the presence of flavonoids. Other authors consider inhibition of tyrosinase due to other chemical constituents [33]. The study suggests the leaves of M. speciosa an alternative to commercially available Arbutin and Vitamin C as the extracts showed significant effect at comparatively lower concentrations. This is the first study to report anti-tyrosinase activity of various extracts of M. speciosa leaves, suggesting its possible use in cosmetics for skin whitening and treatment of hyperpigmentation.

4.4. Sunscreen Activity

During the last few years, human activities has damaged the stratosphere and resulted in a significant ultraviolet (UV) radiations reaching the earth. As a result, various diseases and disorders have been increased alarmingly due to constant exposure to solar UV radiations [34]. The UV radiations have been categorized into three regions i.e. UV-C (200-280nm), UV-B (280-320nm) and UV-A (320-400nm). Among these, UV-C can cause severe damage to skin and eyes for even a short exposure. However, these radiations are completely filtered and absorbed by atmosphere. UV-A and UV-B radiations are not completely absorbed and responsible for sunburn and other skin related disorders [35]. So to avoid such problems, sunscreen agents are used for protection against harmful radiations. Although, synthetic sunscreens are available in the market but with realization of their side effects, herbal based photoprotective agents are more likely to be preferred. In this study, we investigated various extracts of M. speciosa leaves for sunscreen activities in different regions. All the extracts exhibited sunscreen activities almost in all zones of radiations. However, the EtOAc extract showed maximum absorbance in UV-A and UV-B zone as compared to other two extracts. While moderate activities were noted for the PE and MeOH extracts. The sunscreen activity of plant based products is thought to be due to the presence of flavonoids, polyphenols and alkaloids [34,36]. Further study is suggested to screen these classes of compounds to identify the sunscreen compounds, hence it will be a cheaper and safe alternative to harmful synthetic chemicals used in sun-protection cosmetics.

4.5. Anticancer Activity

Breast cancer is the most common life threatening cancer amongst women. According to International Agency for Research on Cancer (WHO) report 2018, 1 in 4 new cancer cases diagnosed in women globally are breast cancer [37]. During the last few years, researchers have started venture into natural bioactive compounds that could possibly lead to comparatively efficient and safe therapeutics to synthetic drugs. Hence, many plants were investigated and reported to show significant potential against breast cancer cells [38]. Different classes of compounds in plant extracts, such as polyphenols, alkaloids and terpenoids have been reported to demonstrate anticancer activities [39].

For the first time, various extracts of M. speciosa leaves were investigated for anticancer properties. From the studies conducted, it may be concluded that the PE extract contained phytocompounds that are responsible for stronger cytotoxic effect against breast cancer cell lines. The results were in agreement to Gunji et al. who reported toxic effects of the PE extract of Cynodon dactylon against MCF-7 cells [40]. The EtOAc extract exhibited moderate cytotoxicity leading to death of cancer cells by almost 57%. These results may be supported by studies conducted by Mardina et al. who reported anticancer activities of the EtOAc extract of Sphagneticola trilobata (L.) J.F. Pruski on MCF-7 cells line [41]. The in vitro studies of the MeOH extract also showed potential for anticancer activity which was in favor of Kamalanathan and Natarajan who reported the anticancer potential of the MeOH extract of leaf and leafderived callus of Aerva javanica [42].

These preliminary results suggest the promising anticancer potential of the PE, EtOAc and MeOH extracts of M. speciosa leaves that might be an ideal choice to develop novel plant-based anticancer drugs.

4.6. Statistical Analysis

All the experiments for bioactivities were run in triplicate. The results were expressed as means± SD (standard deviation). The data was analyzed using one-way analysis of variance (ANOVA) at α = 0.05 with SPSS 22.0 software.

5. Conclusion

The screening performed on extracts of the leaves of M. speciosa for phytochemical composition, antityrosinase, sunscreen and anticancer properties reveals that they are endowed with potentially exploitable natural bioactive compounds and may therefore be suggested as a source of natural medicines. Further, isolation and purification of the active compounds, and in vivo evaluation of bioactivities, along with toxicity studies of the extracts from M. speciosa leaves are therefore suggested.

Acknowledgments

This work was supported by the Key Research and Development Project of Hainan Province (ZDYF2019165), the Innovative Research Team Project of Ministry (IRT-16R19), and the National Natural Science Foundation of China (Nos. 21662012 and 41866005).

References

[1]   | Parthasarathy, S., Juzaili, A., Ramanathan, S., Ismail, S., Sasidharan, S., Said, M. I. M and Mansor, S.M. (2009). Evaluation of antioxidant and antibacterial activities of aqueous, methanolic and alkaloid extracts from Mitragyna speciosa (Rubiaceae Family) leaves. Molecules. 14, 3964-3974.
[2]   | Mariana, V., Eduardo, M. C., Sara, S and Manuela, P. (2020). Impact of plant extracts upon human health: A review. Crit. Rev. Food Sci. Nutr. 60(5): 873-886.
[3]   | Ekor, M. (2013). The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front. Pharmacolog. 4: 177.
[4]   | Enzo, A. P. (2011). Traditional medicinal plant extracts and natural products with activity against oral bacteria: Potential application in the prevention and treatment of oral diseases. Evid. Based complement. Alternat. Med. Article ID 680354, pp. 15.
[5]   | Nasiruddin., Yu, Z., Zhao, T., Chen, G and Minghui, J. (2020). Allelopathic and Medicinal plant. 26. Millettia speciosa. Allelopathy J. 51(1): In press.
[6]   | Candra, I., Foliatini., Hanafi., Lilis, S. and Maman, S. (2018). Volatile compound analysis using GC-MS, phytochemical screening and antioxidant activities of the husk of “Julang-Jaling” (Archidendron bubalinum (Jack) I.C Nielsen) from Lampung, Indonesia. Pharmacogn. J. 10(1): 92-98.
[7]   | Mustapha, N. A and Runner, R. T. M. (2009). GC-MS analysis and preliminary antimicrobial activity of Albizia adianthifolia (Schumach) and Pterocarpus angolensis (DC). Medicines. 3(3): pp. 9.
[8]   | Zhao, L-J., Wei, L., Xiong, S-H., Jie, T., Lou, Z-H., Xie, M-X., Xia, B-H., Lin, L-M and Liao, D-F. (2018). Determination of total flavonoids contents and antioxidant activity of Ginkgo biloba leaf by near-infrared reflectance method. Int. J. Anal. Chem. pp. 7.
[9]   | Park, K. M., Kwon, K.M and Lee, S.H. (2015). Evaluation of the antioxidant activities and tyrosinase inhibitory property from mycelium culture extracts. Evid. Based complement. Alternat. Med. Article ID 616298, pp. 7.
[10]   | Varsha, G., Pooja, B and Pournima, S. (2018). Anti-solar study of ethanolic extract of leaves Moringa oleifera. Int. J. Adv. Community Med. 1(3): 08-10.
[11]   | Varsha, G., Tulashiram, L and Shobha, H. (2108). Anti-solar study of ethanolic extract of leaves Cassia fistula. J. drug deliv. Ther. 8(5-s): 232-234.
[12]   | Maqsood, M., Qureshi, R., Ali, S., Ikram, M., Khan, J. A. (2015). Preliminary screening of methanolic plant extracts against human rhabdomyosarcoma cell line from salt range, Pakistan. Pak. J. Bot. 47(1): 353-357.
[13]   | Valentina, R., Sandra, S. A., Maria, E. M., Diego, U., Johanny, A., Jenny, P. Q and Loango, N. (2019). Biological activity of Passiflora edulis f. Flavicarpa ethanolic leaves extract on human colonic adenocarcinoma cells. J. Appl. Pharm. 9(02): 064-071.
[14]   | Fong, J., Kamola, K., Yaxal, A., Pavel, K., Savo, L., Arkady, M., and Lothar, L. (2015). A novel class of ruthenium-based photosensitizers effectively kills in vitro cancer cells and in vivo tumors. Photochem. Photobiol. Sci. 14(11): 2014-2023.
[15]   | Fawzy, G. A., Areej, M. A and Shagufta, P. (2014). Anticancer activity of flavane gallates isolated from Plicosepalus curviflorus. Phocog. Mag. 10(39): 519-523.
[16]   | Feoktisova, M., Geserick, P and Leverkus M. (2016). Crystal violet assay for determining viability of cultured cells. Cold Spring Harb. Protoc. 343-346.
[17]   | Idowu, J. S., Ayuk, E. O., Elizabeth, K and Wilfred, O-M. (2020). Phytochemical screening and gas chromatographymass spectrometry analysis of ethanol extract of Scambiosa columbabria L. Pharmacogn. Res. 12(1): 35-39.
[18]   | Sundarraj, S., Ramar, T., Sreevani, V., Kaveri, K., Gunasekaran, P., Achiraman, S and Kannan, S. (2012). gamma-sitosterol from Acacia nilotica L. induces G2/M cell cycle arrest and apoptosis through c-Myc suppression in MCF-7 and A549 cells. J. Ethnopharmacol. 141(3): 803-809.
[19]   | Lokesh, R and Kannabiran, K. (2017). Cytotoxic potential of n-hexadecanoic acid extracted from Kigelia pinnata leaves. Asian J. Cell Bio. 12(1): 20-27.
[20]   | Lakshmi, M and Bindu, R. N. (2017). GC-MS analysis of the chloroform extract of bark of Terminalia travancorensis Wight & Arn. (Combretaceae). Int. J. Pharm. Sci. Res. 8(2): 794-98.
[21]   | Mika, D. T and Robert, V. C. (2019). The potential physiological role of γ-tocopherol in human health: A qualitative review. Nutr. Cancer. 72(5): 808-825.
[22]   | Soodabeh, S., Azadeh, M., Ahmad, R. G and Mohammad, A. (2014). The story of beta-sitosterol: A review. European J. Med. Plants 4(5): 590-609.

[23]  
Tareq, A. M., Farhad, S., Uddin, A. B. M. N., Hoque, M., Nasrin, M. S., Uddin, M. M. R., Mohiminul, H., Arafat, S., Munira, M. S., Chadni, L., Hossen, S. M. M., Reza, A.S.M. A and Emran, T. B. (2020). Chemical profiles, pharmacological properties, and in silico studies provide new insights on Cycas pectinata. Heliyon. 6(6), e04061.

[24]  
Adnan, M., Chy, M. N.U., Kamal, A. T. M. M., Chowdhury, K. A. A., Rahman, M. A., Reza, A. S. M. A., Muniruzzaman, M., Rony, S. R., Nasrin, M. S., Azad, M. O. K., Park, C. H., Lim, Y. S and Cho, D. H. (2020). Intervention in neuropsychiatric disorders by suppressing inflammatory and oxidative stress signal and exploration of in silico studies for potential lead compounds from Holigarna caustica (Dennst.) Oken leaves. Biomolecules. 10(4): 561.

[25]   | Kim, B-R., Hyun, M. K., Chang, H. J., Kang, S-Y., Kim, J-B., Yeo, G. J., Kong, Y. P., Lee, I-S and Han, A-R. (2020). Composition and antioxidant activities of volatile organic compounds in radiation-bred Coreopsis cultivars. Plants. 9(6): 717.
[26]   | Mary, A. P. F and Giri, R. S. (2016). Phytochemical screening and GC-MS analysis in ethanolic leaf extracts of Ageratum Conyzoides (L.) World J. Pharm. Res. 5(7): 1019-1029.
[27]   | Sushant, A., Manoj, K. B., Krisha, D., Puspa, K., Roshani, G and Niranjan, K. (2019). Total phenolic content, flavonoid content and antioxidant potential of wild vegetables from western Nepal. Plants. 8(4): 96.
[28]   | Mohammad, A. H., Afaf, M. W and Ahmed, S. H. I. (2019). Comparison of total phenols, flavonoids and antioxidant activity of various crude extracts of Hyoscyamus gallagheri traditionally used for the treatment of epilepsy. Clin. Phytoscience. 5(1): 20.
[29]   | Kumar, V and Roy, B.K. (2018). Population authentication of the traditional medicinal plant Cassia tora L. based on ISSR markers and FTIR analysis. Sci. Rep. 8, 10714.
[30]   | Thanigaimalai, P., Manickam, M and Vigneshwaran, N. (2017). Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. J. Enzyme Inhib. Med. Chem. 32(1): 403-425.
[31]   | Ando, H., Kondoh, H., Ichilhashi, M and Hearing, V. J. (2007). Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase. J. Invest. Dermatol. 127(4): 751-761.
[32]   | Wilfred, O-M and Idowu, J. S. (2020). Anti-Melanogenesis, antioxidant and anti-tyrosinase activities of Scabiosa columbaria L. processes. 8: 236.
[33]   | Adria, F., Guillermo, C., Francisco, L., Eva M. T., Montserrat, M and Victor, L. (2019). Evaluation of anti-tyrosinase and antioxidant properties of four fern species for potential cosmetic applications. Forests. 10: 179.
[34]   | Mayuri, T. N., Benthota, M. A. S. M., Subasinghe, A. K. A., Mohomed, M. Q and Lalith, J. (2016). Photoprotective potential in some medicinal plants used to treat skin diseases in Sri Lanka. BMC Complement Altern. Med. 16:479.
[35]   | Priyanka, S., Mary, S. R. I., Nandini, H. S., Kutty, A. V. M and Kiranmayee, P. (2018). A pilot study on sun protection factor of plant extracts: an observational study. Asian J. Pharm. Clin. Res. 11(4): 67-71.
[36]   | Radhika, B., Neha, S., Ramya, N and Nooreen, N. (2019). Evaluation of sunscreen activity of Hibiscus Hirtus Linn. J. Bio. Innov. 8(2): 127-133.
[37]   | International Agency for Research on Cancer, WHO. Geneva, Switzerland. (2018). Latest global cancer data: Cancer burden rises to 18.1 million new cases and 9.6 million cancer deaths in 2018.
[38]   | Joanna, J. L., Latifah, S. Y., Kassim, N. K., Abdullah, C. A. C., Nurulaidah, E., Lim, P. C and Tan, D. C. (2020). Cytotoxic activity of Christia vespertilionis root and leaf extracts and fractions against breast cancer cell lines. Molecules. 25: 2610.
[39]   | Roy, A., Jauhari, N and Bharadvaja, N. (2018). Medicinal plants as a potential source of chemopreventive agents. In Anticancer Plants: Natural products and biotechnological implements; Akhtar, M.S., Swamy, M.K., Eds.; Springer: Singapore. 109-139.
[40]   | Gunji, V., Swarupa, R. T., Vani, M and Vineela, P. A. J. (2015). In-vitro anticancer activity of petroleum ether extract of Cynodon dactylon. J. Pharmacogn. Phytochem. 4(1): 164-168.
[41]   | Mardina, V., Ilyas, S., Harmawan, T., Halimatussakdiah, H and Tanjung, M. (2020). Antioxidant and cytotoxic activities of the ethyl acetate extract of Sphagneticola trilobata (L.) J.F. Pruski on MCF-7 breast cancer cell. J. Adv. Pharm. Technol. Res. 11(3): 123-7.
[42]   | Kamalanathan, D and Natarajan, D. (2020). Anticancer potential of leaf and leaf-derived callus extracts of Aerva javanica against MCF-7 breast cancer cell line. J. Can. Res. Ther. 14:321-7.