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A Study on Physicochemical Properties and Antimicrobial Activities of Phytochemical Constituents Extracted from the Stem Bark of Neolamarckia cadamba (Roxb.) Bosser (Ma-u)

Nay Soe Moe1, Aung Kyaw Swar2, Saw Hla Myint3 Abstract This research deals mainly with studies on physicochemical properties and antimicrobial activities of the different crude extracts such as water (H2O), ethanol (EtOH), ethyl acetate (EtOAc) and petroleum ether (PE) of the stem bark of Neolamarckia cadamba (Roxb.) Bosser (Ma-u). The sample was collected from Maubin Township, Ayeyarwady Region. The antimicrobial activities of Ma-u were screened by using agar well diffusion method against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumilus, Candida albicans and Escherichia coli species. In addition, the qualitative and quantitative determination and elemental analysis of the selected sample were determined by phytochemical tests. The soluble matter contents of various organic solvents such as ethanol (5.90 %), methanol (8.07 %), pet-ether (0.31 %), ethyl acetate (1.22 %) and water (9.25 %) were observed. According to the experiments, the moisture (10.68 %), ash (7.82 %), protein (7.35 %), crude fiber (28.80 %), crude fat (1.64 %), carbohydrate (43.71 %) and calories (219 kcal/ 100 g) were found in the bark of Neolamarckia cadamba. The relative abundance of elements in the bark of Neolamarckia cadamba was determined by EDXRF technique which reveals that Ca (5.422 %), K (3.691 %), S (0.412 %) and C (90.289 %) were observed as major constituents. Keywords: Neolamarckia cadamba (Roxb.) Bosser (Ma-u), soluble matter contents, phytochemical tests, physicochemical properties, antimicrobial activities

Introduction Research in medicinal plants has gained a renewed focus recently. The main reason is that the other systems of medicine are associated with a number of side effects and can cause serious problems. There has been a number of flora in use for medicinal purposes over past several centuries. Varieties of plants have been used for medicinal treatments, either whole or specific parts (bark, root, leaves, fruit, flowers, seeds) in the dried state. Now they are formulated into suitable preparations such as tablets, pills, extracts, tinctures, lotions, ointments and creams. Neolamarckia cadamba (Roxb.) Bosser (Ma-u) is an evergreen tropical tree widely grown in Australia, China, India, Indonesia, Malaysia, Philippines, Singapore, Vietnam, Bangladesh, Nepal, Myanmar and Sri Lanka. The Cadamba has been known to cure a number of diseases, particularly, the extract prepared from the bark and leaves is crucial. Various researchers across the world have focused their studies on discovering a number of phytochemicals as well as secondary metabolites with pharmacological significance from the Cadamba. (Dwevedi, A., et al., 2015)

1 Lecturer, Department of Chemistry, Maubin University 2 Lecturer, Department of Chemistry, University of Yangon 3Part-time Professor, Department of Chemistry, University of Yangon

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Outstanding Features Trees, deciduous, to 30 m tall; trunk with small buttresses; bark thin, grayish brown, fissured and scabrous when old; branches horizontally spreading, flattened becoming subterete, glabrescent. Petiole 20–35 mm, glabrous; leaf blade drying thinly leathery, elliptic or oblong-elliptic, adaxially shiny and glabrous, abaxially glabrous to densely puberulent, base shallowly cordate on juvenile growth, rounded or truncate on adult growth, apex acute; secondary veins 8–12 pairs; stipules lanceolate, acute. Inflorescences flowering heads with peduncle, rather stout. Calyx puberulent to pilosulous; ovary portion ellipsoid to obovoid, partially to deeply lobed; lobes oblong to spatulate, obtuse to rounded. Corolla yellowish white, funnelform, outside glabrous. Fruiting heads yellowish green, 30–40 mm in diam., with peduncles markedly thickened. Fruit cylindrical to ellipsoid or obovoid, glabrous; seeds 3angled. (Kress, W.J., et al., 2003 and Wu, Z.Y., et al., 1994) Botanical Aspects of Selected Sample Kingdom : Planate Family : Rubiaceae Botanical name : Neolamarckia cadamba (Roxb.) Bosser Genus : Neolamarckia Species : cadamba English name : Bur-flower tree Myanmar name : Ma-u, Ma-u-let-tan-she Part used : Stem bark Chemical Constituents and Health Benefits of Neolamarckia cadamba (Roxb.) Bosser Studies on discovering a number of phytochemical constituents of Neolamarckia cadamba showed saponins, indole and quinoline alkaloids, secoindoids, triterpenes, kaempferol and ursolic acid with pharmacological significance. The various extracts have been known to cure a number of diseases; particularly, the extracts prepared from the bark and leaves is crucial. The commercially available compounds from cadamba were cadambagenic acid, quinovic acid, E-sitosterol, cadambine, cadamine, etc., so cadamba plant can produce a lot of biologically active compounds. (Dwevedi, A., et al., 2015) According to traditional medicine in India, the bark and leaves have great significance of biological and pharmacological activities such as analgesic, antipyretic, anti-inflammatory, antifungal, diuretic and laxative, hypolipidemic, antioxidant, antidiabetic, antihepatotoxic, antimalarial, antihelminthic, antivenon and antitumor. (Gautam, R., et al., 2012)

Materials and Methods

Sample Collection and Botanical Identification of Collected Sample The samples were randomly collected in Maubin Township, Ayeyarwady Region in August, 2017. The collected samples were identified as Neolamarckia cadamba (Roxb.) Bosser according to the authorized botanist at Department of Botany, Maubin University.

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Figure 1 Stem bark, leaf and flowers of Neolamarckia cadamba (Roxb.) Bosser Sample Preparation The collected cadamba samples were washed with water and dried at room temperature. The dried samples were ground into purely fine powder by using an electric grinder and then stored in air-tight container to prevent moisture changes and other contaminations. Extraction of Matter Contents in the Stem Bark of Neolamarckia cadamba (Roxb.) Bosser The extractable matter contents (1:20 w/v) will be determined with various organic solvents such as ethanol, methanol, pet-ether, ethyl acetate and water. 2 g of dry powder sample were taken into each of five conical flasks of 150 mL. 40 mL of each EtOH, MeOH, PE, EtOAc and H2O were added to each flask. Then, the flasks were shaken continuously for 24 hours and allowed to stand for 24 hours. The supernatant solution was filtered by the filter paper (< 2 μm pore size). The filtrate was dried, weighed and the percent yield was calculated. Phytochemical Investigation for Classification of Compounds The phytochemical constituents were qualitatively determined by using Dragendorff’s/Mayer’s reagent, ninhydrin, Benedic’s solution, 10% lead acetate, 5% FeCl3, 70% HCl, concentrated H2SO4, 10% NaOH, 1% CuSO4 and acetic anhydride. Quantitatively, total alkaloid content was determined by Harborne’s method (1984), total glucose-related carbohydrate content was determined by Phenol- sulphuric acid method and total saponin content was determined by Obadoni and Ochukos’ method (2001). Determination of Physicochemical Parameters of Stem Bark Sample The moisture content of dried powdered samples was determined by oven dry method. The crude protein content was determined by micro-Kjeldahl method. The crude fat content was determined by Soxhlet extraction method. The fiber content was determined by digestion method. The total carbohydrate content was determined by subtraction method (the difference between 100 and the sum of the percentages of ash, fat, fibre, moisture and protein).The percentage of ash was obtained by ashing method. The energy value of samples was calculated by usual equation. Energy value (kcal/100g) = (4 × protein %) + (4 × carbohydrate %) + (9 × fat %) Determination of Elemental Analysis of Stem Bark Sample The elemental contents (Relative abundance) in the stem bark of Neolamarckia cadamba (Roxb.) Bosser were determined by EDXRF technique. Screening of Antimicrobial Activity of Cadamba Stem Bark For the examination of vitro antimicrobial activity, solvent extracts such as PE, EtOAc, EtOH, MeOH and H2O of the cadamba stem bark were investigated by using agar well diffusion method. Then, the extracts were tested against six pathogenic microorganisms (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumilus, Candida albicans and Escherichia coli) by using agar well diffusion method. Maubin University Research Journal 2020, Vol.11 165

Determination of Glucose-related Carbohydrate Content of Selected Sample Glucose-related carbohydrate contents of two different extracts (H2O and EtOH) were determined from absorbance at 490 nm by using PD-303UV spectrophotometer. The concentrations were measured by two-fold dilution. Results and Discussion The phytochemical tests revealed that alkaloids, α-amino acids, carbohydrates, flavonoids, glycosides, phenolic compounds, proteins, reducing sugars, steroids, saponins, tannins and terpenoids were present in Neolamarckia cadamba (Roxb.) Bosser (Ma-u). Their positive and negative quantities are observed in Table 1. The presence of secondary metabolites such as alkaloids, flavonoids, glycosides, steroids, saponins, tannins and terpenoids in the plant sample can contribute to their medicinal values. The percentages of extractable matter contents in the bark sample are shown in Table 2 and Figure 2. The physicochemical parameters such as moisture, ash, protein, crude fiber, crude fat, carbohydrate and calories were determined in the stem bark of Neolamarckia cadamba. The results are shown in Table 3 and Figure 3. From these results, it was found that crude fibre and carbohydrate were present as major nutrients in samples. Fiber can prevent and treat constipation, help to lower the risk of hemorrhoids and assist the reduction of blood pressure. Carbohydrates are compounds of carbon, hydrogen and oxygen. Almost all of the dietary carbohydrates are eventually utilized to meet the energy requirements of the body. Proteins are complex nitrogenous substances of high molecules which are found as components of all living matters and contain a large number of amino acids linked together. Protein is required for growth and developments as well as maintenance and repair. Table 1 Screening on the Phytochemical Constituents by Using the Different Extracts of Neolamarckia cadamba (Roxb.) Bosser (Ma-u) Stem Bark Samplep

(+) = present, (-) = absent 166 Maubin University Research Journal 2020, Vol.11

Table 2 Extracted Matter Contents in the Stem Bark of Neolamarckia cadamba (Roxb.) Bosser

No. Solvent Weight (w/w %)

1 Ethanol 5.90

2 Ethyl acetate 1.22

3 Methanol 8.07

4 Petroleum ether 0.31

5 Water 9.25

EtOAc

Figure 2 Histogram of soluble matter contents in the stem bark of Neolamarckia cadamba (Roxb.) Bosser

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Table 3 Physicochemical Parameters of Stem Bark of Neolamarckia cadamba (Roxb.) Bosser Sr Test Parameter Test Method Result No. (%) 1. Moisture Oven drying method 10.68 2. Ash Muffle furnace method 7.82 3. Protein Macro-Kjeldahl distillation 7.35 4. Crude Fiber Acid and alkali digestion method 28.80 5. Crude Fat Soxhlet extraction 1.64 Carbohydrate Subtraction method 43.71 6. 7. Energy value (kcal / 100 g) Energy value equation 219

Figure 3 Histogram of physicochemical parameters in the stem bark of Neolamarckia cadamba (Roxb.) Bosser

Some major elements such as calcium, potassium, sulphur and carbon were observed in the stem bark of Neolamarckia cadamba. Some trace elements such as phosphorus, silicon, iron, manganese, copper and zinc were present in the stem bark. The results are shown in Table 4 and Figure 4.

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Table 4 Relative Abundance of the Predominant Elements in Neolamarckia cadamba Stem Bark Sample by EDXRF

No. Elements Relative Abundance (%)

1 Calcium 5.422

2 Potassium 3.691

3 Sulphur 0.412

4 Carbon 90.289

Figure 4 EDXRF spectrogram of Neolamarckia cadamba (Roxb.) Bosser (Ma-u)

In the antimicrobial investigations, all extracts except PE show the antimicrobial activity. The antimicrobial activities of the different crude extracts (H2O, MeOH, EtOH, EtOAc, and PE) were screened by using agar well diffusion method against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumilus, Candida albicans and Escherichia coli species. The activities of MeOH extract on the microorganisms are considerably high (zone of inhibition ranged 26 mm). It can be obviously seen that PE extract of Neolamarckia cadamba did not exhibit the potent antimicrobial activity. EtOH and EtOAc extracts had inhibition zone ranging between 14 to 25 mm testing with all species of microorganisms. The results obtained from the tested samples are shown in Table 5 and Figure 5. Maubin University Research Journal 2020, Vol.11 169

Table 5 Screening of Antimicrobial Activities on the Phytochemical Constituents by Using the Different Extracts of Neolamarckia cadamba (Roxb,) Bosser Stem Bark Sample

Diameter of inhibition zone (mm) for Neolamarckia cadamba (Roxb.) Bosser (Ma-u) Organisms used PE MeOH EtOH EtOAc H2O extract extract extract extract extract

(1) B.subtilis - 25 (+++) 14 (+) 20 (+++) 20 (+++)

(2) S.Aureus - 20 (+++) 25 (+++) 20 (+++) 17 (++)

(3) P.aeruginosa - 20 (+++) 23 (+++) 20 (+++) 15 (++)

(4) B.Pumilus - 26 (+++) 25 (+++) 18 (++) 16 (++)

(5) C. albicans - 25 (+++) 14 (+) 21 (+++) 15 (++)

(6) E.coli - 30 (+++) 25 (+++) 18 (++) 18 (++)

SampleSample Control SampleSaammple CoControlntrol Samplep CoControlntrol

Sample Control SampleSaSample CoControlntrol Sample Control Sample CoControlntro Bacillus pumilus

Figure 5 Antimicrobial screening of crude extracts of Neolamarckia cadamba (Roxb.) Bosser (Ma-u)

Different concentrations of 0.20, 0.40, 0.60, 0.80 and 1.00 mg/mL of standard glucose solution were prepared to calibrate their absorbance and concentrations as shown in Table 6. The calibration curve of standard glucose solution from absorbance at 490 nm is shown in Figure 6. In the detection of glucose-related carbohydrate content, two 170 Maubin University Research Journal 2020, Vol.11

polar solvent extracts, H2O and EtOH were used and the results were be observed in Table 7.

Table 6 Absorbance at 490 nm with Different Concentrations of Standard Glucose Solution

Blank 1 2 3 4 5

Absorbance 0 0.008 0.011 0.013 0.015 0.018

Concentration (mg/mL) 0 0.20 0.40 0.60 0.80 1.00

Figure 6 Calibration curve of standard glucose solution at 490 nm

Table 7 Glucose-related Carbohydrate Content of Two Different Extracts of Sample from Absorbance at 490 nm

Concentration (mg/mL) No. Extract Absorbance (Two-fold dilution)

1 EtOH 0.103 8.10

2 H2O 0.126 10.02

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Conclusion

From the overall results of present research work, there was a significant presence of saponins, flavonoids, alkaloids, steroids, glycosides, terpenoids and carbohydrates in Neolamarckia cadamba. The different extracts (EtOH, MeOH, PE, EtOAc and H2O) yield the respective phytochemical constituents, mainly secondary metabolites which are used as medicines, flavorings, pigments and recreational drugs. The secondary metabolites, alkaloids and glycosides, were present in all extracts. These secondary metabolites show pharmacological activities (analgesic, antiasthma antimalarial, antipyretic, antitumor, antiarrhythmic, anticholinergic, heart failure and certain irregular heartbeats etc). Flavonoids, phenolic compounds, steroids, saponins, tannins, and terpenoids are also used as medicines (as in antioxidants, antimicrobial, antidiabetic etc.) Flavonoids exhibit various pharmacological effects including antioxidant, anti- inflammation, anti-platelet, anti-allergic, cytotoxicity, reduce risk for heart disease or cancer etc. Apart from tanning, tannins are also used in dyeing, photography, refining beer and wine as well as an astringent in medicines. Saponins have many beneficial effects on blood cholesterol levels, cancer, bone health and stimulation of the immune system. Antimicrobial activities reveal that MeOH extract is more potent than the other extract although PE extract of Neolamarckia cadamba did not exhibit the potent antimicrobial activity. In addition, total alkaloid (0.0528 %), total carbohydrate content (43.71%) in which glucose-related carbohydrate contents are (8.10 mg/mL) in EtOH extract and (10.02 mg/mL) in H2O extract and total saponin (5.8200 %) indicate confidentially that Neolamarckia cadamba (Roxb.) Bosser (Ma-u) Stem Bark have illustrated as a source of some active organic compounds. Acknowledgements

Special thanks are due to Dr Aung Kyaw, Rector of Maubin University for providing the full facilities and his permission to submit this research paper. I extend my profound gratitude to Dr Win Myint Thein and Dr Lwin Lwin Myint, Pro-rectors, Maubin University for their keen interest and also for their permission. I would like to express my sincere gratitude to Dr Hnin Hnin Than, Professor and Head, Department of Chemistry, Maubin University for her encouragement and valuable suggestion throughout this research work. I would also like to express my profound gratitude to Dr Than Than Khaing, Professor, Department of Chemistry, Maubin University for her kind help and support of this research paper. I am also thankful to Dr Saw Hla Myint, Part-time Professor, Department of Chemistry, University of Yangon for his close supervision and Dr Aung Kyaw Swar, Lecturer, Department of Chemistry, University of Yangon for his guidance and advice during this research work.

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