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RESEARCHORIGINAL ARTICLE ARTICLE PEER REVIEWED | OPEN ACCESS Chemical composition and testosterone level enhancing property of oblonga leaf essential oil in male albino rats

Frank N I Morah, Emmanuel A Bisong

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Aim: Sterculia oblonga is an African medicinal Morah FNI, Bisong EA. Chemical composition and used to enhance male fertility. The aim of testosterone level enhancing property of Sterculia the present study is to determine the chemical oblonga leaf essential oil in male albino rats. Edorium J composition and testosterone level enhancing- Public Health 2019;6:100024P16FM2019. property of its leaf essential oil in male albino rats. Methods: Fresh leaves of S. oblonga were ground and steam-distilled to get its essential Article ID: 100024P16FM2019 oil. The individual constituents of the essential oil were separated by gas chromatography and ********* identified by mass spectrometry. The rats were fed with different doses of the essential oil for doi: 10.5348/100024P16FM2019RA one week. The level of testosterone in their blood serum after one week was determined using ultraviolet (UV)-spectrophotometer at wave length of 450 nm. Results: The essential oil contains 33 constituents and it increased INTRODUCTION testosterone level in the blood serum of the Sterculia oblonga is a tropical timber which albino rats. Conclusion: The essential oil contains is also employed in herbal medicine. Different natural 33 constituents which are being identified for products have been reported in Sterculia species. For the first time in S. oblonga. The essential oil example, Sterculia foetida contains lutedin, quercetin, exhibited testosterone level enhancing property anthocyanins, coumarins, and hydroxybenzoic acid [1, 2]. which is dose dependent in male albino rats. There is scanty report on the chemical constituents of S. oblonga. The oven dried wood contains cellulose, 36.2– Keywords: Chemical composition, Essential oil, Ster- 46.3%; furfural, 11.5–12.4%; pentosans, 19.8–21.2%; culia oblonga, Testosterone level lignin, 18.2–22.7%, and ash, 1.3–3.2% [3]. It also contains tannins, 1240 mg/100 g; alkaloids, 10%; flavonoids, 3.4%; oxalate, 1.31 g/100 g; phenols, 620 mg/g; cyanide, Frank N I Morah1, Emmanuel A Bisong2 1.66 mg/100 g, and lipid, 4.8% [4]. 1 Affiliations: PhD, Professor of Natural Product Chemistry, Sterculia oblonga is used traditionally in parts Department of Chemistry, University of Calabar, Calabar, of and Southern to control male Cross River State, Nigeria; 2MSc, Lecturer in Organic Chem- istry, Department of Chemistry, University of Calabar, Cala- infertility but there is no report on its biological bar, Cross River State, Nigeria. activity. Increase in serum testosterone level on drug administration is by enhancing the production of Corresponding Author: Frank N I Morah, Chemistry De- luteinizing hormone, enhancing the viability of Leydig partment, University of Calabar, Calabar, Cross River State, Nigeria; Email: [email protected] cells, reducing testicular oxidative injury, enhancing StAR gene [5]. The present work is therefore aimed at determination of the chemical constituents and effect Received: 03 June 2019 of S. oblonga leaf essential oil on the level of serum Accepted: 30 July 2019 testosterone in male white albino rats. Published: 03 September 2019

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MATERIALS AND METHODS Six microplate wells for the essential oil and seventh one for control were used. Appropriate serum reference Fresh S. oblonga leaves were harvested from Ikot w (10 mL) was pipetted and assigned into the wells. 50 mL Omini, Calabar, Cross River State, Nigeria. It was of the working testosterone enzyme reagent was added. authenticated by Frank Adeoye of the Herbarium unit, The microplates were swirled gently for 30 seconds to Botany Department, University of Calabar while white mix. They were covered and incubated for 60 minutes at albino rats were procured from the animal house of the room temperature. The contents of the microplates were Physiology Department, University of Calabar. discarded by decantation and blotted dry with absorbent The fresh leaves of S. oblonga (800 g) were rinsed with paper. A 350 mL of washing buffer was added and decanted. distilled water, crushed and immediately steam distilled This was repeated two times to make a total of three for three hours. The steam distillate was collected in a washes. Manual plate washer was employed. A 100 mL of separatory funnel. The lower aqueous layer was run off working substrate solution was added. All the reagents while the upper essential oil layer was collected. were added in the same order to all the wells to minimize The constituents of the essential oil were separated reaction time between different wells. The mixture was by gas chromatography while the individual constituents incubated for 15 minutes at room temperature. Finally, were identified by mass spectrometric analysis [6]. The 50 mL of stop solution was added to each well and gently compound identification was done by comparison of the mixed for 20 seconds. The absorbance in each well was obtained mass spectra with those of the standard mass read at 450 nm within 30 minutes of addition of the spectra of organic compounds from National Institute of stop solution. A calibration graph of absorbance versus Standard and Technology (NIST) library [6]. concentration was used for determination of testosterone Nine white albino rats each weighing 150 g were level. selected from the animal house. Another set weighing 160 g each were also selected for the work. The essential oil was administered orally at a dose of 1.6 g/kg of body weight for RESULTS the 150 g rats and a dose of 3.0 g/kg body weight for the 160 g rats. The oil was administered twice daily for a period Table 1 shows that S. oblonga leaf essential oil has 33 of one week after which they were sacrificed to obtain their constituents, 25 of which are oxygenated. It also shows blood. The blood was allowed to clot for the serum sample the relative molecular mass, formula, and structure of to be obtained. The specimens were centrifuged to separate these compounds. Table 2 shows that the essential oil the serum from the blood cells. Table 1: Gas chromatography-mass spectroscopy analysis of essential oil from Sterculia oblonga S/N Compound name Retention Molecular Relative Base Percentage Chemical time formula molecular peak composition structure (minutes) mass 1 p-xylene 7.433 C H 106 91 0.59 8 10

2 2-Heptenal, (z)- 11.129 C H O 112 83 1.23 7 12

3 Octanal 13.100 C8H16O 128 57 0.60 O 4 4,5-Dimethyl-hept-2- 16.784 C H OH 142 71 1.18 9 17 en-3-ol OH

5 Nonanal 17.166 C H O 142 57 1.68 9 18 O

H 6 Octanoic acid 20.412 C H O 144 60 1.11 8 16 2

O

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Table 1: (Continued)

S/N Compound name Retention Molecular Relative Base Percentage Chemical time formula molecular peak composition structure (minutes) mass 7 2-sec- 22.427 C H O 154 55 4.76 10 18 Butylcyclohexanone O

8 2-Decenal, (z)- 22.946 C10H18O 154 70 4.76 O

9 2,4-Decadienal, (E,Z)- 24.040 C H O 152 81 2.58 10 16 O

10 trans-Undec-4-enal 24.197 C11H20O 168 55 1.170 (E,E)- O

11 2, 4-Decadienal, (E,E)- 24.822 C10H16O 152 81 7.60 O

12 2-Undecenal 26.155 C11H19O 167 70 5.99 O

13 n-Decanoic acid 26.887 C10H20O2 172 68 1.04 O

OH

14 Dodecanoic acid, methyl 31.397 C13H26O2 226 74 4.29 O ester O

15 Dodecanoic acid 33.204 C12H24O2 224 73 18.99 O

OH

16 Dodecanoic acid, ethyl 33.511 C14H28O2 228 88 0.86 O ester O

17 Hexadecane 33.636 C16H34 226 57 0.71

18 tert-Hexadecanethiol 36.176 C16H34S 258 97 0.92 SH

19 Methyl tetradecanoate 36.564 C H O 242 74 1.30 15 30 2 O O

20 Tetradecanoic acid 37.152 C14H28O­ 2 228 73 4.36 O

OH

21 Tetradecanoic acid, ethyl 37.370 C19H32O2 256 88 0.71 O ester O

22 Octadecane 37.414 C18H38 254 57 0.86

23 Diethylene glycol 38.209 C11H23 CO2 288 57 1.50 O O monolaurate C2H2OC2H2OH O OH

24 Hexadecanoic acid, 38.484 C15H31CO2CH3 270 74 3.41 methyl ester

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Table 1: (Continued) S/N Compound name Retention Molecular Relative Base Percentage Chemical time formula molecular peak composition structure (minutes) mass

25 tert-Hexadecanethiol 38.584 C35H33SH 258 57 0.62 SH

26 Hexadecanoic acid 38.815 C15H31CO2H 256 73 6.85 O

OH H 27 Hexadecanoic acid 38.947 C15H31COOC2H5 284 88 2.23 O ethyl ester O

28 (E)-Pentatriacont-17-ene 39.297 C35H70 490 57 0.66

29 10-Octadecanoic acid, 39.585 C17H33CO2CH3 296 55 7.85 O methyl ester O

30 cis-13-Eicosenoic acid 39.973 C18H35CO2H 296 57 2.02

O HO

31 (Z)-Pentatriacont-17-ene 40.811 C35H70 490 57 2.37

32 cis-13-Eicosenoic acid 42.350 C18H35CO2H 296 55 2.12

O HO

33 Diisooctyl phthalate 43.188 C6H4(CO2 364 149 3.76 O O O C8H17)2 O

Table 2: Effects of dose of Sterculia oblonga essential oil on testosterone level in male albino rats Dose g/kg body weight % Mortality Testosterone level (ng/cm3) 1.6 g/kg 0% 0.65± 0.3

3 g/kg 0% 0.74 ± 0.1 0 g/kg 0% 0.57 ± 0.1 caused rise in testosterone level in the albino rats’ blood of these compounds are fatty acids. Dodecanoic acid (i.e., serum and no death occurred after seven days of oral lauric acid) is mainly used for soap production. In vivo administration of the essential oil. It also shows that experiments have shown that lauric acid and some other increase in the dose of the oil administered resulted in fatty acids are used for treatment of acne [7, 8]. further increase in the serum level of testosterone. None of the rats died after seven days of administration of the essential oil. This shows that the S. oblonga leaf essential oil is not toxic to the rats. At the daily dose of 1.6 g/ DISCUSSION kg of body weight, the level of testosterone increased from 0.57 to 0.65 ng/cm3 in control and sample respectively. The constituents of the essential oil include At a daily dose of 3 g/kg of body weight testosterone level dodecanoic acid (18.99%), 2-sec-butylcyclohexanone increased from 0.57 in the control to 0.74 ng/cm3 in the (4.76%), 2Z-decenol (4.70%), 2,4-decadienal E, Z blood serum after seven days of administration. This (2.58%), methyldodecanoic acid (4.29%), 2,4-decadienal, further shows that the effect of S. oblonga leaf essential E,E-(7.6%), tetradecanoic acid (4.36%), n-hexadecanoic oil on testosterone level is dose dependent. Since the level acid (6.58%), diisooctyl phthalate (5.76%), and of testosterone is directly related to the degree of male 10-octadecanoic acid (7.8%). All these compounds are fertility [9, 10], the essential oil increases fertility in the being identified for the first time in S. oblonga. A number male rats. This gives credence to the use of S. oblonga

Edorium Journal of Public Health, Vol. 6; 2019. Edorium J Public Health 2019;6:100024P16FM2019. Morah et al. 5 www.ejpublichealth.com to correct male infertility. The use of herbs in health 11. Morah FNI, Apebende GC. Chemical composition, care delivery generally has the advantage of having less anthelmintic and antimicrobial activity of the hazardous side effect and is environmentally friendly essential oil of the aerial part of Cleome rutidosperma. [11]. Although one may be tempted to hold the major International Journal of Advanced Scientific Research 2018;3(4):48–54. constituents responsible for the observed biological 12. Morah FNI, Ashipu LB. Chemical composition and activity, such activities are known to be modulated by the antimicrobial activity of essential oil from Heinsia minor constituents which may have synergistic, additive, crinite leaf. American Journal of Essential Oil and and antagonistic effects [12]. Natural Products 2017;5(2):23–8.

********* CONCLUSION Author Contributions The S. oblonga leaf essential oil contains 33 natural Frank N I Morah – Conception of the work, Design of the products which are being reported for the first time in work, Acquisition of data, Analysis of data, Interpretation S. oblonga. These compounds are responsible for the of data, Drafting the work, Revising the work critically observed blood serum testosterone level enhancing for important intellectual content, Final approval of the activity which is dose dependent. Since it is not toxic to version to be published, Agree to be accountable for all the rats, it could serve as an alternative source of male aspects of the work in ensuring that questions related anti-infertility drug. to the accuracy or integrity of any part of the work are appropriately investigated and resolved REFERENCES Emmanuel A Bisong – Conception of the work, Design of the work, Acquisition of data, Analysis of data, 1. Xia PF. Study on the chemical constituents and Interpretation of data, Drafting the work, Revising the bioactivities of Sterculia foetida L. Peking Union work critically for important intellectual content, Final Medical College, China 2009. approval of the version to be published, Agree to be 2. Xia PF, Feng ZM, Yang YN, Zhang PC. Two flavonoid accountable for all aspects of the work in ensuring that glycosides and a phenylpropanoid glucose ester from questions related to the accuracy or integrity of any part the leaves of Sterculia foetida. J Asian Nat Prod Res of the work are appropriately investigated and resolved 2009;11(8):766–71. 3. Bosh CH, Louppe D. Stercliaoblonga mast. In: Louppe Guarantor of Submission D, Oteng-Ainoako AA, Brink M, editors. Plant Research of Tropical Africa 7(1): Timbers 1. Wageningen, The corresponding author is the guarantor of submission. Netherlands: PROTA Foundation; 2008. 4. Ezeonu CS, Ejikeme CM. Qualitative and quantitative Source of Support deteramination of phytochemical contents of None. indigenous Nigerian softwoods. New Journal of Science 2016;9. Consent Statement 5. Banihani SA. Mechanisms of honey on testosterone Written informed consent was obtained from the patient levels. Heliyon 2019;5(7):e02029. for publication of this article. 6. Morah FNI, Apebende GC. Chemical composition, anthelmintic and antimicrobial and activity of the chloroform fraction of ethanol extract of Conflict of Interest Cleome rutidosperma. Edorium J Public Health Authors declare no conflict of interest. 2018;5:100019P16FM2018. 7. Nakatsuji T, Kao MC, Fang JY, et al. Antimicrobial Data Availability property of lauric acid against Propionibacterium All relevant data are within the paper and its Supporting acnes: Its therapeutic potential for inflammatory acne Information files. valuris. J Invest Dermatol 2009;129(10):2480–8. 8. Yang D, Pornpattananangkul D, Nakatsuji T, et al. Copyright The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes. Biomaterials © 2019 Frank N I Morah et al. This article is distributed 2009;30(30):6035–40. under the terms of Creative Commons Attribution 9. Crosnoe LE, Grober E, Ohl D, Kim ED. Exogenous License which permits unrestricted use, distribution testosterone: A preventable cause of male infertility. and reproduction in any medium provided the original Transl Androl Urol 2013;2(2):106–13. author(s) and original publisher are properly credited. 10. Omisanjo OA, Ikuerowo SO, Abdulsalam MA, Please see the copyright policy on the journal website for Ajenifuja SO, Shittu KA. Use of exogenous more information. testosterone for the treatment of male factor infertility: A survey of Nigerian doctors. Int J Reprod Med 2017;2017:4607623.

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