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Quality evaluation, GC/MS analysis and antimicrobial activities of Morinda Citrifolia against oral Microorganisms

Suparat Jongjai1, Jongkon Saising2, Rawiwan Charoensub1, 3, Pravaree Phuneerub1, 3*

1Department of Applied Thai Traditional Medicine, School of Integrative Medicine, Mae Fah Luang University, Chiang Rai 57100, Thailand. 2School of Health Science, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand. 3Medicinal Plants Innovation Center of Mae Fah Luang University, Mae Fah Luang University, Chiang Rai 57100, Thailand.

Correspondence: Pravaree Phuneerub, Department of Applied Thai Traditional Medicine, School of Integrative Medicine, Mae Fah Luang University, Chiang Rai 57100, Thailand. [email protected]


ABSTRACT

In Thai traditional textbooks, Morinda citrifolia. fruit is an antidote to vomiting and cure oral diseases. The current study aimed to develop the standardization parameters, chemical evaluation, and anti-microorganisms activities of M. citrifolia fruit extracts against oral microorganisms including Staphylococcus aureus, Streptococcus mutans, and Candida albican.  M. citrifolia fruit (MC) were collected from 15 sources throughout Thailand and then examined the pharmacognostic specification including macroscopic characteristics, microscopic characteristics, and physicochemical parameters. The volatile oils of Morinda citrifolia fruit (MCO) were determined by GC/MS. The antimicrobial activity of ethanol extract (MCE), water extract (MCW), and volatile oils (MCO) of M. citrifolia fruit were carried out by examining the inhibition areas using the agar well diffusion method, minimal inhibition concentration (MIC), and minimal bactericidal concentration (MBC). The outcome of the study revealed that Physico-chemical identification showing a loss on drying, total ashes, acid-insoluble ashes, and water content should be not more than 10.992, 10.115, 0.973, and 6.882% w/w, respectively. The volatile oil content, water, ethanol, and hexane soluble extractive values should not be less than 0.762, 31.8257, 18.0992, 3.4964 % w/w, respectively. GC-MS analyses of MCO showed the major compound was octanoic acid (84.103%) and hexanoic acid (7.183%). Based on antimicrobial activities, MCE and MCW cannot have antimicrobial activity in the oral cavity. However, MCO was potentially effective in suppressing microbial growth agiant S.aureus and S.mutans, and C. albicans. The volatile oil of M.citrifolia fruit may be a beneficial component of oral health care products or drugs for oral disease.

Keywords:  Morinda citrifolia, Antimicrobial activities, Pharmacognostic specification, Chemical analysis


Introduction  

The disadvantaged and poor population groups in both developing and developed countries still have problems about the burden of oral disease, for instance, periodontal disease, dental caries, tooth loss, oral mucosal lesions, and oropharyngeal cancers which had a great impact on general health and life quality [1]. The significance of bacteria strain is the of oral microorganisms including Enterobacterfaecalis, Lactobacillus fermentum, Lactobacillus salivarius, Streptococcus sobrinus, and Streptococcus mutans, and all of bacteria are Gram positive and cause cariogenic [2]. Streptococcus mutans are bacteria that are commonly found in the mouth on the surface of teeth and that are the main cause of dental caries in humans. S. aureus (gram-positive bacteria) and C. albicans (opportunistic fungal) are the cause of denture stomatitis (DS) and can perform biofilm formation [3].  In addition, Candida spp is to be important in the initiation of Oral candidiasis which is a common opportunistic infection of the oral cavity. Nowadays, antibiotics such as vancomycin, teicoplanin (glycopeptide), and linezolid (oxazolidinone) are used to fight this bacterium. However, they have limited available and adverse effects [4].

Morinda citriolia L. is an evergreen shrub or small tree. It is from the Rubiaceae family. It is growing in tropica regions worldwide. It is known as Noni and in Thai called “Yor ban” or “Yor” [5].  M. citrifolia is largely used in traditional medicine in primary health care to relieve illness. In Thai traditional textbooks it is stated that the unripe fruit is an antidote to vomiting, the ripe fruit is a woman’s menstrual drive and unripe fruit burned into charcoal is mixed with a little salt which is used for treatment swollen gums and gum diseases. In previous studies, M. citrifolia extract had significant property concern with antibacterial and antifungal activity against various strains of bacteria and fungi. The ethanol extract of dried noni fruit  have displayed significant antibacteria activity against foodborne pathogens such against S.typhimurium and E. coli [6]. M.citrifolia fruit showed antioxidant, anti-inflammatory, liver-protective, and immunomodulatory effects [7]. However, there are no reports about pharmacognostic specification of M. citrifolia fruit and lack of evidence about antimicrobial activities against oral microorganisms. Therefore, the purpose of this research is to develop the standardization parameters of M. citrifolia crude drug, chemical analysis, and antimicrobial activities against oral microorganisms from MCE, MCW, and MCO to scientific evidence for supporting the use of M. citrifolia extract as an alternative treatment in oral diseases.

Materials and Methods    

Plant materials 

Dired MC samples were gathered from 15 different locations in 5 parts of Thailand during August-September 2020 (North, Central, North-East, East, and South) and they were authenticated by the office of the Queen Sirikit National Convention, ChiangMai, Thailand. Hot air oven at 50°C was used to dry the samples and then they were peverized for further investigation.

Plant extraction

Grinded the dried M.citrifolia fruit (MC) from the Supan Buri province of good standardized quality control were macerated with 99% ethanol and water. MC (5 g) were continuously extracted with 99% ethanol until exhaustion using soxhlet apparatus. The extract was filtered and evaporated to be dried under a vacuum. MC (100 g) were continuously macerated with DI water until exhaustion, and then Whatman No. 4 filter paper was used to filter the extract. The water extracts were lyophilized to be dried and stored at -20 °C until being used to reduce the probability of degradation of active compounds.

Determination of macroscopic and microscopic characteristics

Macroscopic specifications of herbal materials indicate shape, size, color, surface characteristics, texture, fracture characteristics, and appearance of the cut surface and were illustrated by drawing and photograph of the plant. Microscopic characteristics were used for identification and authentication of plant material by cross-section, powder drug by microscope under an OLYMPUS (CX22LED) magnifying lens (4X to 40X), and expressed by drawing and photograph of the plant by the researcher [8].

Physico-chemical specifications 

Loss on drying, total ash, acid-soluble ash, water content, and solvent extractive values were following by World Health Organization guidelines of the Quality control methods for medicinal plant materials [8].

Determination of volatile oil content

50 gr of powder was poured into round bottom flasks, then 500 ml of distilled water and boiling chips were added and boiled in Clevenger type apparatus for 6 hrs. and volume of the volatile oils was collected and recorded [8]. Each sample was done in three replications. The volatile oil from Supan Buri was used for GC/MS analysis and antimicrobial activities.

Gas chromatograph mass spectrometer (GCMS)

The volatile oil dissolves in hexane. GC/MS analysis was determined by Agilent 6890 GC with HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thicknesses). The oven temperature was ramped from 60°C to 325 °C at a constant rate of 3 °C per minute. The injection port was held 220°C. The gas carrier in this testing was Helium gas with a flow rate of one milliliter per minute and then obtained, analyzed, and compared the mass spectra index of mass spectral of NIST Mass Spectral Library.

Preparation of microbial strains and growth conditions

Microbial organisms including S. aureus (ATCC 25923), S. mutans (DMST 1877), and C. albicans (ATCC  10231) were collected from STIC, Mae Fah Luang University. C. Albicans were cultivated in Brain Heart Infusion Agar (BHA) media and in the dish. And then, they were nurtured for 24 hrs at 37°C. S. mutans and S.aureus were cultured on Trypticase soy agar (TSA) and Mueller Hinton Agar (MHA), respectively. S.aureus was  incubated at 37°C for 24 hrs and S. mutans was  incubated at 37°C for 48 hrs. And then, they were adjusted turbidity equal to McFaLand No. 0.5, then diluted until colony to have approximately to about 1.0×106  CFU/ml.

Agar disc diffusion method

Disc diffusion method is generally used to investigate the antimicrobial activity of plants or microbial extracts. This test of antibacterial by agar disc diffusion was adapted from the suggested guidelines and protocols from the Clinical and  Laboratory  Standards  Institute (CLSI). Sterilized Whatman papers discs (6 mm diameter) were soaked with 10 µl of MCE (2mg/disc), MCW (2mg/disc), MCO (2mg/disc), chloramphenicol (30μg/disc), candixclotrimazole (0.1mg/disc) as positive control, DMSO and tween 20 as negative control.  Three organisms was spread on plates by swabed C.albicans cultures on BHA plate, S. mutans cultured and S. aureus cultured on MHA. And then, discs were added. The plates were incubated at 37°C for 18-24 hrs. The diameters of inhibitory zones were measured in millimetres by Vernier caliper [9].

 

Specification of MIC, MBC, and MFC

The least inhibitory condensation was specified by broth microdilution technique n 96 well microtiter plate according to the clinical and laboratory standards institute (CLSI). The MCO, chloramphenicol, candixclotrimazole (were dissolved in tween 20), and then dilutions series were prepared in a 96-well plate, ranging from 0.5 µg/ml to 32 µg/ml. 100 μl of extracts or a positive/negative control in broth and 100 μl of inoculate broth were poured into each well and incubated at 37°C, for 24 hours. The least inhibitory condensation was seen at the last well which was indicated in a clear solution. Streaked clear inoculate broth on Mueller Hinton agar (for bacteria) and Sabouraud Dextose - agar (for fungi) then nurtured the agar plate for 24 hours at 37 °C. Minimum bactericidal concentration (MBC) and minimum fungicidal concentrations (MFC) of the extract were investigated from the agar plate with no emerged microbial expansion [10].

 

Statistical analysis

The Physico-chemical identification was offered in the Grand mean ± pooled SD values. Antimicrobial activities were done in triplicates and findings are offered as the mean ± standard deviation (SD).

 

 

b)

c)

a)

d)

Figure 1. Macroscopic evaluation a) Drawing of Composition of M.citrifolia  Leaves and Cross-section of Fruit, b) Fresh  fruit, c) Transverse section of fruit, d) Dried fruits of M.citrifolia

 

Figure 2. Cross-section of M.citrifolia Fruit: Thin-Walled Epidermis (1), Collenchyma (2), Parenchyma Cells (3), Prismatic Calcium Oxalate (4), Resin (5), Oils (6), Raphide Calcium Oxalate Crystals (7), Bundle of Spiral Vessel (8), Pith Parenchyma (9)

 

Figure 3. Microscopic characteristics of M.citrifolia Fruit Was Powder: Reticulated Vessel (a), Parenchyma (b), Oils (c), Prismatic Calcium Oxalate Crystals (d), Longitudinal Parenchyma Wit Oil Gland (e), Oils in Fiber Group (f), Oils with Parenchyma (g), Calcium Oxalate Crystals (Raphide) (h)

 

 

Table 1. Physicochemical Evaluation of M.citrifolia Fruit

Parameter % (by weight)

Mean ± SD*

Maximum - Minimum

Loss on drying

10.992 ± 0.337

17.776 - 5.990

Total ash

10.115 ± 1.812

11.849 - 8.321

Acid-insoluble ash

0.973 ± 0.161

1.244  - 0.708

Volatile oil content

0.762 ± 0.095

1.399 - 0.199

Water content

6.882 ± 1.159

9.326  - 2.832

Ethanol-soluble extractive

18.099 ± 0.668

23.889 -3.789

Hexane-soluble extractive

3.496 ± 0.439

4.460 - 0.0002

Water-soluble extractive

31.826 ± 2.946

37.519 - 17.214

*Grand mean±pooled SD. The specimen were from 15 various sources throughout Thailand, and each sample was done in triplicate. SD: Standard Deviation

 

Table 2. The Chemical Constituents of the Volatile Oil of M.citrifolia Fruit Using GC/MS

RT

Chemical compounds

Molecular Formula

Molecular weight

Area%

4.3653

3-Methyl-3-buten-1-OL

C5H10O

86.13

0.056

8.9921

2-Heptanone

C7H14O

114.19

0.047

10.3651

Methyl hexanoate

C7H14O2

130.1849

0.367

14.0224

Hexanoic acid

C6H12O2

116.16

7.183

14.7836

2-Pentyne

C5H8

68.12

0.050

18.9745

Butanoic acid, 2-methyl-, 3-methyl-3-butenyl ester

C10H18O2

170.25

0.091

19.5085

Methyl octanoate

C9H18O2

158.24

2.177

24.3414

Octanoic acid

C8H16O2

144.21

84.103

25.8362

Isobutyl 3-methylbut-3-enyl carbonate

C10H18O3

186.25

0.927

27.346

Hexanoic acid, 3-methyl-2-butenyl ester

C11H20O2

184.2753

0.0928

28.631

Decanoic acid, methyl ester

C11H22O2

186.2912

0.105

30.7668

n-Decanoic acid

C10H20O2

172.26

1.072

30.9297

[Dodecanoyl(methyl)amino]acetic acid

C13H25NO3

243.3420

0.124

34.3094

Succinic acid, isobutyl 3-methylbut-3-enyl ester

C14H22O4

254.32

2.790

35.6283

Octanoic acid, 3-methylbut-2-enyl ester

C13H24O2

212.33

0.277

39.1784

Hexyl octanoate

C14H28O2

228.37

0.058

42.0099

Dodecanoic acid, 4-penten-1-yl ester

C17H32O2

268.4

0.058

51.2587

Hexadecanoic acid, methyl ester

C17H34O2

270.4507

0.131

56.507

9,15-Octadecadienoic acid, methyl ester, (Z,Z)-

C19H34O2

294.25588

0.165

56.6972

9-Octadecenoic acid, methyl ester, (E)-

C19H36O2

296.4879

0.099

62.2251

9-Octadecenamide

C18H35NO

281.5

0.028

 

Results and Discussion

M.citrifolia is a small perennial with 2-6 meters in height. Leaves are simple leaves, opposite, with ample large elliptical leaves (5-17 cm length, 10-40 cm width), fresh green young leaves. Flowers are little tubular, white, banded together, and inserted on the peduncle. The petioles leave ring-like marks on the stalks and the corolla is greenish-white. Fruit (3-10 cm length, 3-6 cm width) is ovular and fleshy with an embossed appearance were showed in Figure 1. It is somewhat rugged, semi-translucent, and ranges in color from green to yellow to almost white at the time of picking (Figure 1).  It is covered with little reddish-brown buds including the seeds [11].  The pulp is juicy and bitter, a light dull yellow or whitish color, and gelatinous. Ripe fruit has many hard triangular reddish-brown pits which are found, each including four seeds (approximately 3.5 mm) [12]. Microscopic characteristics of cross-section and powder of crude drug of MC were illustrated in Figures 2 and 3. Microscopic powder features revealed the presence of oil glands, raphide, and prismatic calcium oxalate crystals that were in agreement with the earlier report by Pratima and Shrikanthin in 2015 [13].

Physicochemical evaluation plays an important role in detecting the purity and quality of crude drugs including loss on drying, total ash, acid insoluble ash, volatile oil content, extractive values, and water content parameters. The determination of ash is useful for detecting inorganic materials such as metallic salts, silica, carbonates, calcium oxalate crystals, sandy in crude drug [14]. The physicochemical specifications (% by weight) of the fruit of M.citrifolia fruit are demonstrated in Table 1. They are evaluated from 15 sources in Thailand. The loss on drying, total ash, acid insoluble ash, volatile oil content, and water content should not be more than 10.992, 10.115, 0.973, and 6.882% w/w respectively. The water, ethanol, and hexane soluble extractive values should not be less than 0.762, 31.826, 18.099, 3.496 % w/w, respectively.

The volatile oil by hydrodistillation method of M.citrifolia fruit detected, contained 21 components (Table 2). The major chemical constituents revealed that octanoic acid (84.103%) and hexanoic acid (7.183%) has similarities with previously research [15-17]. In previous study [18] demonstrated that octanoic acid and hexanoic acid showed a strong effect on inhibition of growth of againt E. coli and S. aureus. According to Bae and Rhee in 2019 [19] have stated that the caprylic acid showed antifungal effects against C. albicans. In 2020, Halala et al. and Hsieh et al. appraised the effect of the capric acid alone and indicated a stronger effect against oral Candida isolates than its combination with common antifungal drugs [20, 21].

The bacteria strain of oral microorganisms S. mutans and S. aureus as Gram-positive bacteria are the main cause of dental caries and parotitis in humans [22, 23]. Besides, C. Albicans is a fungus that is the main causative agent of oral candidiasis [24]. The MCE, MCW, and MCO were investigated the antimicrobial activity by agar disc diffusion method with S. aureus and S. mutans, and C. Albicans. Evaluation of the antibacterial activity of M. citrifolia fruit extract was recorded in Table 3 and Figure 4. The results revealed that MCE and MCW extract was not potentially effective in suppressing microbial growth of the oral cavity. However, MCO has potentially inhibited the growth of S. aureus and S. mutans and C. albicans with inhibition zones of 13, 9, and 19 mm, respectively. The minimum inhibitory concentration results, bacterial MICs, and minimal fungicide concentration, MFC with C. Albicans showed 2 and 4 µg/ml, respectively. MCO demonstrated strong antimicrobial activity against C. Albicans. Similarly, a previous study reported by Luis et al. [25]  suggested the oil of M. citrifolia fruit had potential antifungal activity against fungi C. Albicans.

 

 

Table 3. Antimicrobial Screening Test of M.citrifolia Fruit Extracts

Extract

S. aureus

S.mutans

C.albicans

Inhibition zones (mm)

MIC

(µg/ml)

MBC

(µg/ml)

Inhibition zones (mm)

MIC

(µg/ml)

MBC

(µg/ml)

Inhibition zones (mm)

MIC

(µg/ml)

MFC

(µg/ml)

MCO

13

32

32

9

4

32

19

2

4

CP

27

4

16

24.6

2

4

-

-

-

CA

-

 

 

-

-

-

9.6

2

4

MCO: Volatile Oil of M. citrifolia Fruit CP: Chloramphenicol CA: Candixclotrimazole

 

Figure 4. Zone of Inhibition of Oral Organisms of Volatile Oil of M.citrifolia Fruit (o: Volatileoil of M. citrifolia Fruit, DM : (DMSO) Negative Control, and ca (Candix Clotrimazole): Positive Control

Conclusion

The major component of volatile oils of M.citrifolia fruit (MCO) was octanoic acid and hexanoic acid. According to the results obtained in this study for antimicrobial activities, MCO showed effectiveness in suppressing microbial growth in the oral cavity including gram-positive bacteria, S.aureus and S.mutans, and indicated strongly inhibition to the growth of the fungus is C.albicans. It can be further developed into a drug for the alternative treatment of oral candidiasis and maybe a beneficial component of oral health care products.

Acknowledgments: The authors are grateful to Mae Fah Luang University for funding and providing laboratory facilities throughout the work. This work was supported by providing laboratory facilities throughout the work from the Medicinal Plants Innovation Center of Mae Fah Luang University Thailand. In addition thanks to Miss Mesa Nuansalee, Miss Apisamai Duangrasri, Miss Jintapa Srisuwan, and Mr. Russalee Jaroenrit who supported the team in all experiments.

Conflict of interest: None

Financial support: This work was supported by research funding from the Medicinal Plants Innovation Center of Mae Fah Luang University, Mae Fah Luang University.

Ethics statement: None

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