Formulation, characteristics and anti-bacterial effects of Euphorbia hirta L. mouthwash.

Objective: Mouthwash is a liquid solution used to improve oral health and breath freshness as well as reduce oral bacteria. This study aims to formulate a Euphorbia hirta L. ethanol extract for mouthwash, evaluate the physical properties, and determine its anti-bacterial effects against Streptococcus mutans.
Methods: Each mouthwash formula was created by utilising a solubilisation technique. Three mouthwash formulas were created from different concentrations of Euphorbia hirta L. ethanol extract (0.5%, 1%, and 2%), and referred to as F1, F2, and F3. The organoleptic properties, pH levels, specific gravity, viscosity, flow properties, stability, irritation level, contact time, and anti-bacterial effects were evaluated for each concentration. Result: The resulting formulas featured a distinctive smell of oleum menthae piperitae and had a sweet and spicy taste. These characteristics remained unaffected during storage. The acidity levels ranged from 4.59 to 6.0, the weight masses ranged from 0.9693 to 1.0710 g/ml, and the viscosity ranged from 1.50 to 3.00 cP. F3 concentration was non-irritating with mucus production at 11.325%, had lower contact time with a neutralisation of 57.14% of Streptococcus mutans colonies in 30 seconds, and showed stable anti-bacterial control against Streptococcus mutans (p = 0.000) within the first week of use.
Conclusion: The study results offer the first proof of Euphorbia hirta L. used for improving human health. The formulation features the ideal physical and stability characteristics of mouthwash, and possesses anti-bacterial properties that can potentially combat Streptococcus mutans. Clinical use of the formulated mouthwash must be explored in future research.

Introduction

Bacteria found on the surface of teeth, especially in plaque, can cause several types of dental caries, such as Streptococcus mutans, Streptococcus sobrinus, Streptococcus downei, and Staphylococcus aureus. Streptococcus mutans is a common type of flora found in the oral cavity and dominates the composition of bacteria in plaque. Toothbrushes, interdental cleaners, mouthwash, and antimicrobial agents can be used to prevent plaque formation. Chemical substances used in mouthwash have antiseptic or anti-bacterial properties to inhibit plaque formation. Mouthwash is used to improve oral health and aesthetics, reduce bacteria, remove bad odours and prevent dental caries. Moreover, mouthwash can reach areas in the mouth that may be difficult to reach with a toothbrush.

The use of herbal products as medicine is increasing in popularity, and a growing body of research is focused on the use of medicinal plants in Indonesia, particularly medicinal plants used to improve oral health. Herbal mouthwash products can be easily found in the Indonesian market. Indonesia is particularly known for its indigenous medicinal plants and has a long history of producing natural products. Euphorbia hirta L. (also known as Patikan Kebo) is a traditional medicinal plant widespread in Indonesia and used to treat conjunctivitis and skin, gastrointestinal, bronchial and respiratory diseases. The leaves of Euphorbia hirta L. are traditionally ground and used in rural areas of Indonesia as an antiseptic solution and as medicine to reduce itching. These leaves are used to prepare tea for the treatment of asthma. Euphorbia hirta L. is a wild plant that typically grows on the surface of semi-dry soil and can be found throughout Indonesia. Currently, this plant is not receiving sufficient attention from the public, even though it has considerable potential for medicinal uses. Euphorbia hirta L. can be used to treat various diseases because it contains alkaloid chemical compounds, tannins, polyphenol compounds, and flavonoids.

Euphorbia hirta L. can be used to inhibit the formation of biofilms, such as Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Salmonella typhi, Proteus vulgaris, Proteus mirabilis, and Streptococcus pyogenes. Research has noted that Euphorbia hirta L. can inhibit the growth of specific types of Gram-positive bacteria, such as Staphylococcus aureus, Micrococcus sp., Bacillus subtilis, and Bacillus thuringensis. It also inhibits the growth of certain types of Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumonia, Salmonella typhi, and Proteus mirabilis, and inhibits fungal growths, such as Candida albicans, with inhibition zones ranging from 16 to 29 mm.

Therefore, determining the effectiveness of Euphorbia hirta L. for anti-bacterial preparation for oral mucosa treatment is necessary. The purpose of this study is to evaluate the physical properties and characteristics of Euphorbia hirta L. extract and determine its anti-bacterial effects against Streptococcus mutans.

Materials and Methods

Euphorbia hirta L. sample

Euphorbia hirta L. samples were collected from the Panam area in the subdistrict of Tampan, in the district of Simpang Baru, in Pekanbaru, Indonesia. Mature plants were collected in polythene bags, and the samples were identified in a botanical laboratory (Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Riau). The samples were assigned the registration number 66/UN/19.5.1.1.3/Bio/Botani/2019 (see Figure 1).

Figure 1

Euphorbia hirta L or Patikan kebo (A) and sample specimen (B).

Euphorbia hirta L. extraction

A total of 3500 grams of Euphorbia hirta L. was used to create an ethanol extract. It was cleaned, dried at room temperature for 24 hours, and processed in a blender until its texture became smooth.

To obtain the extract from the processed Euphorbia hirta L., 2000 ml of a 96% ethanol solution was used, and this process was repeated thrice. The initial extract was filtered and evaporated at temperatures ranging from 50 °C to 60 °C to obtain a 100% pure Euphorbia hirta L. extract. The pure extract was weighed, stored in a sealed glass container, and subsequently placed in a desiccator before being used as mouthwash.

Euphorbia hirta L. mouthwash formulation

As much as 100 ml of mouthwash was produced for each formulation with Euphorbia hirta L. extract as the active substance. The formulations of Euphorbia hirta L. mouthwash (referred to as F1, F2, and F3) are presented in Table 1.

Table 1

Euphorbia hirta L mouthwash formulation.

Component	Formulation (%)
	FI	F2	F3
Ethanolic extract of Euphorbia hirta L	0.5	1	2
Propylene glycol (CV.Clorogreen)	25	25	25
Tween 80 (CV.Clorogreen)	5	5	5
Oleum menthe piperitae (CV.Clorogreen)	0.25	0.25	0.25
Benzoate acid (CV.Clorogreen)	0,1	0,1	0,1
Sodium benzoate (CV.Clorogreen)	1	1	1
Sorbitol 70% (CV.Clorogreen)	15	15	15
Aquadest	ad 100	ad 100	ad 100

Propylene glycol was included in the Euphorbia hirta L. extract and placed in a glass beaker. It was then raised to 60 °C, stirred with a magnetic stirrer at 300 rpm and Tween 80, and sorbitol and aquadest were added.

Benzoic acid and sodium benzoate were dissolved in aquadest and added to the solution and stirred with a magnetic stirrer until homogeneous. Subsequently, 100 ml of the sorbitol qs and aquadest ad was stirred until the solution became clear, and Oleum menthae piperitae was added.

Organoleptic testing

Three Euphorbia hirta L. mouthwash formulations were tested by random respondents. A questionnaire was administered to record observations on colour, smell, and taste and denote significant differences in preparation methods. Organoleptic testing was performed on the same respondents once a week for a duration of eight weeks.

Acidity (pH)

The pH testing was conducted with a Hanna pH meter once a week for eight weeks, and the pH meter was calibrated using a buffer solution (pH 4 and pH 7). The electrodes were rinsed with distilled water and dried. Measurements were noted while preparing each Euphorbia hirta L. mouthwash in a beaker.

Stability test

Stability testing was conducted with the freeze-thaw test and carried out on the Euphorbia hirta L. mouthwash formulations in six cycles. Each cycle was observed after 48 hours of storage at 4 °C and again after 48 hours at 40 °C, and this process continued for 24 days.

Weight mass

The weight mass of each Euphorbia hirta L. mouthwash preparation was measured once per week for eight weeks using a Pyrex pycnometer. The pycnometer was cleaned with distilled water and dried before each use.

Viscosity test

Viscosity was measured using a Brookfield viscometer. Before measuring, the tool was adjusted by levelling the spindle boundary found on the surface of the tool. Thereafter, 100 ml of Euphorbia hirta L. mouthwash was immersed to the specified spindle limit mark. The viscometer was turned on for ±10 seconds, the size was set, and the instrument was turned off. Viscosity was calculated by the viscosity value on the specified spindle scale.

Irritation test

A slug mucosal irritation test was conducted on each of the three Euphorbia hirta L. mouthwash formulations and a control solution containing no Euphorbia hirta L. extract. The snails were weighed, placed on a 1 g sample of mouthwash preparation and left for 60 minutes. The snails were then removed from the preparation and weighed once again. The percentage of mucus production indicates the rate of irritation and is demonstrated by the following formula:

Contact time

Contact times of the three Euphorbia hirta L. mouthwash formulations were measured in sterile test tubes by adding 100 μL of Streptococcus mutans suspension to 10 ml of each formulation. After 30 seconds of contact, the solution mixtures absorbed as much as 50 μL, and they were placed in a natrium agar medium. They were each rotated three times to the left and three times to the right. The plates were incubated at 37 °C for 18–24 hours, and the results were observed. Each area on the medium that showed the least amount of colony growth with the shortest contact time was determined the most effective. Decrease in bacteria colonies is demonstrated by the following formula:

Anti-bacterial activity

A standard diffusion test was conducted to determine the anti-bacterial effects of each of the Euphorbia hirta L. mouthwash formulations. Anti-bacterial properties of each formulation were measured before and after one week of storage.

Additionally, 300 μL of the isolated Streptococcus mutans (Oxoid, Thermo Scientific) colony was selected and suspended in a natrium agar medium. A total of 15 ml of each of the three Euphorbia hirta L. formulations were added to the medium. Enkasari mouthwash was used as a control group and did not contain the Euphorbia hirta L. extract. After an incubation period of 24 hours at 37 °C, the size of the inhibition zone, which appears as the clearing zone, formed around the paper disc was measured. The test was repeated thrice.

Data analysis

The anti-bacterial effects of Euphorbia hirta L. mouthwash were analysed using one-way ANOVA and post-hoc Tukey HSD tests with p < 0.05, thus showing significant differences in each group with SPSS software.

Results

Organoleptic testing results are shown in Table 2. The F1 solution was yellowish brown, had a distinctive smell of oleum menthae piperitae, as well as a sweet and spicy taste. The F2 solution was brown, had a distinctive smell of oleum menthae piperitae, and a sweet and spicy taste. The F3 solution was dark brown and had a distinctive smell of oleum menthae piperitae.

Table 2

Organoleptics testing of Euphorbia hirta L mouthwash for 8 weeks.

		Week
		1	2	3	4	5	6	7	8
FI	Form	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid
	Smell	OMP	OMP	OMP	OMP	OMP	OMP	OMP	OMP
	Taste	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy
	Color	yellowish brown	yellowish brown	yellowish brown	yellowish brown	yellowish brown	yellowish brown	yellowish brown	yellowish brown
F2	Form	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid
	Smell	OMP	OMP	OMP	OMP	OMP	OMP	OMP	OMP
	Taste	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy
	Color	brown	brown	brown	Brown	brown	brown	brown	brown
F3	Form	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid	Liquid
	Smell	OMP	OMP	OMP	OMP	OMP	OMP	OMP	OMP
	Taste	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy	SweetSpicy
	Color	dark brown	dark brown	dark brown	dark brown	dark brown	dark brown	dark brown	dark brown

OMP = Oleum menthe piperitae

The form, smell, taste, and colour of each solution remained consistent, with no changes after eight weeks of storage.

The results of pH testing are presented in Table 3. The pH levels decreased in each formulation during eight weeks of storage. The pH levels of F1 ranged from 4.79 to 6.05, those of F2 ranged from 4.70 to 5.87, and those of F3 ranged from 4.59 to 5.79.

Table 3

pH of Euphorbia hirta L mouthwash formulation for 8 weeks.

	Week
	1	2	3	4	5	6	7	8
FI	6.05	5.22	5.06	5.04	5.01	5.00	4.88	4.79
F2	5.87	5.16	4.96	4.95	4.93	4.91	4.75	4.70
F3	5.79	4.98	4.78	4.75	4.72	4.72	4.68	4.59

Stability

The freeze-thaw test was conducted on each of the three Euphorbia hirta. L. mouthwash formulations at 4 °C and 40 °C for six cycles and showed no separation.

The weight masses of each the three Euphorbia hirta. L. formulations over an eight-week period are presented in Table 4. F1 ranged from 0.9689 to 1.0641 g/ml, F2 ranged from 0.9693 to 1.0655 g/ml, and F3 ranged from 0.9699 to 1.0710 g/ml.

Table 4

Weight mass of Euphorbia hirta L mouthwash for 8 weeks.

	Week
	1	2	3	4	5	6	7	8
FI	0.9689	1.0274	1.0498	1.0551	1.0576	1.0589	1.0598	1.0641
F2	0.9693	1.0312	1.0504	1.0555	1.0581	1.0593	1.0613	1.0655
F3	0.9699	1.0399	1.0532	1.0569	1.0585	1.0598	1.0623	1.0710

A viscosity test was conducted on each of the three Euphorbia hirta L. formulations after eight weeks of storage, and findings ranged from 1.50 to 3.00 cP (see Table 5).

Table 5

The viscosity of Euphorbia hirta L. mouthwash.

	Rpm	Storage
		before (cP)	after (cP)
F1	20	1,50	3,00
F2	20	1,80	3,00
F3	20	1,80	3,00
control	20	2,10	3,06

F3 had the highest mucus production at 14.965%, followed by F2 at 12.515% and F1 at 11.325%. Mucus production in the positive control was measured at 8.229%, higher than that of the negative control at 5.514%. These results show that Euphorbia hirta L. mouthwash is non-irritating (see Table 6).

Table 6

The irritation test of Euphorbia hirta L. mouthwash.

	Replication	Mucous weight (g)	Snail weight (g)	Mucous (%)	Mean (%)
F1	1	1.173	14.330	12.219	11.325
	2	1.243	12.934	10.430
F2	1	0.925	12.080	13.063	12.515
	2	1.512	18.097	11.967
F3	1	0.934	15.755	16.865	14.965
	2	1.164	15.210	13.064
control (−)NaCl Fisiologis	1	2.494	15.481	6.208	4.514
	2	5.499	15.501	2.819
control (+)Enkasari	1	1.989	15.748	7.918	8.229
	2	1.610	13.747	8.540

Contact time test

Contact testing of Euphorbia hirta L. mouthwash at the lowest dilution (10−5) showed that it can inhibit the growth of Streptococcus mutans colonies in 30 seconds, which is less than the total number of bacterial colonies. Decrease in the number of bacterial colonies in F1, F2 and F3 was 28.57%, 44.76%, and 57.14%, respectively (see Table 7).

Table 7

Contact time evaluation.

	Colonies (CFU/ml)			mean ± SD (CFU/ml)	Colonies decreasing (%)
	1	2	3
Control media	0	0	0	–	–
Control bacteria	105	97	112	105 ± 7.51	–
F1	74	71	79	75 ± 4.04	28.57
F2	60	54	61	58 ± 3.79	44.76
F3	49	41	46	45 ± 4.04	57.14

Anti-bacterial effects of Euphorbia hirta L. against Streptococcus mutans before storage showed a larger inhibition zone in F3 (20.8 ± 0.56), compared with the control negative (7.7 ± 1.46) (p = 0.000). The control positive (24.1 ± 0.51) showed a larger inhibition zone, compared with the control negative (7.7 ± 1.46), F1 (18.6 ± 2.15) and F2 (20.2 ± 0.72) (p = 0.000, p = 0.002 and p = 0.023) (see Figure 2A and Table 8).

Figure 2

Anti-bacterial activity of Euphorbia hirta L mouthwash before storage (A) and after storage (B).

Table 8

Anti-bacterial activity of Euphorbia hirta L mouthwash.

	before storage				1 week after storage
	Zone inhibition (mm)			mean ± SD	Zone inhibition (mm)			mean ± SD
	1	2	3		1	2	3
F1	18.6	20.7	16.4	18.6 ± 2.15	17.5	16.8	17.2	17.2 ± 0.35
F2	20.4	19.3	20.7	20.2 ± 0.72	18.5	17.9	17.2	17.9 ± 0.66
F3	21.4	20.3	20.7	20.8 ± 0.56	21.3	19.9	217	20.9 ± 0.95
Control (−)	8.2	8.9	6.1	7.7 ± 1.46	6.1	7.1	6.7	6.6 ± 0.50
Control (+)	24.6	23.9	23.6	24.1 ± 0.51	23.6	23.2	24.5	23.7 ± 0.67

After a week of storage, the anti-bacterial effects of Euphorbia hirta L. against Streptococcus mutans showed the largest inhibition zone in F3 (20.9 ± 0.95), compared with F2 (17.9 ± 0.66), F1 (17.2 ± 0.35) and the control negative (6.6 ± 0.50) (p = 0.000 p = 0.001 and p = 0.000). The control positive (23.7 ± 0.67) showed the largest inhibition zone, compared with other formulations (p < 0.05) (see Figure 2B and Table 8).

Discussion

The importance of genus Euphorbia is not only represented by its growth forms but also the ability to treat cancer, dengue fever, vitiligo, actinic keratosis, digestive system disorders, infections, respiratory system disorders, and pain. The genus Euphorbia is also used as a catalyst in producing various nanoparticles for medical use.18, 19, 20, 21, 22, 23, 24, 25 Our results highlight the use of natural Euphorbia hirta L. for mouthwash.

This study shows that Euphorbia hirta L. mouthwash has anti-bacterial effects that fight Streptococcus mutans, which is the leading cause of dental caries. The leaves,,26, 27, 28 branches, and aerial part,30, 31, 32, 33, 34, 35 of Euphorbia hirta L. possess anti-bacterial properties. Studies have shown that Euphorbia hirta L. also has anti-bacterial effects against various types of Streptococci, including Streptococcus aureus, Streptococcus faecalis, and Streptococcus pneumoniae., In addition to Streptococcus, the anti-bacterial effects fight against various types of Gram-positive and Gram-negative bacteria (see Table 9).

Table 9

Antibacteria activity of Euphorbia hirta L.

Part of Euphorbia hirta L	Extraction methods	Methods	Antibacterial activity	Reference
Leaves	Methanolic	disc diffusion method	- Inhibit Staphylococcus aureus at concentration 12.50 mg/ml - Inhibit Micrococcus sp at concentration 100 mg/ml - Inhibit Bacillus subtilis at concentration 100 mg/ml - Inhibit Bacillus thuringensis at concentration 100 mg/ml - Inhibit Escherichia coli at concentration 3.13 mg/ml - Inhibit Klebsiella pneumonia at concentration 100 mg/ml - Inhibit Salmonella typhii at concentration 100 mg/ml - Inhibit Proteus mirabilis at concentration 50 mg/ml	14
Leaves	Methanolic	disc diffusion method	Inhibit Pseudomonas aeruginosa	26
	Ethanolic	disc diffusion method
	Aqueous	disc diffusion method
Leaves	Methanolic	agar well diffusion method	- Inhibit Bacillus subtilis at concentration 220 μg/ml - Inhibit Staphylococcus aureus at concentration 220 μg/ml - Inhibit Escherichia coli at concentration 250 μg/ml	27
	Aqueous		Inhibit Escherichia coli at concentration 160 μg/ml
Leaves	Methanolic	Broth microdilution method	- Inhibit Proteus mirabilis - Inhibit Staphylococcus aureus	28
Young branches with leaves and inflorescence	Methanolic	Inhibitory Concentrations in Diffusion (ICD) method	- Inhibit Escherichia coli at concentration 25 μg/ml - Inhibit Pseudomonas aeruginosa at concentration 12.5 μg/ml - Inhibit Proteus vulgaris at concentration 50 μg/ml - Inhibit Klebsiella pneumoniae at concentration 12.5 μg/ml	29
	Aqueous		- Inhibit Escherichia coli at concentration 50 μg/ml - Inhibit Pseudomonas aeruginosa at concentration 25 μg/ml - Inhibit Proteus vulgaris at concentration 100 μg/ml - Inhibit Klebsiella pneumoniae at concentration 12.5 μg/ml
Aerial	Methanolic	tube dilution method	- Inhibit Escherichia coli at concentration 0.189 mg/ml - Inhibit Pseudomonas vulgaris at concentration 1.2 mg/ml - Inhibit Pseudomonas aeruginosa at concentration 0.166 mg/ml - Inhibit Bacillus subtilis at concentration 0.296 mg/ml - Inhibit Bacillus pumilus at concentration 0.269 mg/ml - Inhibit Streptococcus aureus at concentration 0.216 mg/ml - Inhibit Streptococcus faecalis at concentration 0.214 mg/ml	30
Aerial	Methanolic	Broth dilution method	- Inhibit Escherichia coli at concentration 100 mg/ml - Inhibit Klebsiella pneumoniae at concentration 50 mg/ml - Inhibit Shigella dysentriae at concentration 100 mg/ml - Inhibit Salmonella typhi at concentration 50 mg/ml - Inhibit Proteus mirabilis at concentration 50 mg/ml	31
	Aqueous		- Inhibit Escherichia coli at concentration 50 mg/ml - Inhibit Klebsiella pneumoniae at concentration 25 mg/ml - Inhibit Shigella dysentriae at concentration 50 mg/ml - Inhibit Salmonella typhi at concentration 25 mg/ml - Inhibit Proteus mirabilis at concentration 25 mg/ml
Aerial	Ethanolic	tetrazolium microplate assay	- Inhibit Enterobacter aerogens at concentration 1 mg/ml - Inhibit Escherichia coli at concentration 0.5 mg/ml - Inhibit Klebsiella pneumonia at concentration 1 mg/ml - Inhibit Proteus mirabilis at concentration 0.5 mg/ml - Inhibit Proteus vulgaris at concentration 0.25 mg/ml - Inhibit Pseudomonas aeruginosa at concentration 0.125 mg/ml - Inhibit Salmonella typhi at concentration 0.031 mg/ml - Inhibit Shigella dysenteriae at concentration 0.5 mg/ml - Inhibit Staphylococcus aureus at concentration 0.5 mg/ml - Inhibit Bacillus subtilis at concentration 0.25 mg/ml	32
Aerial	Methanolic	tetrazolium microplate assay	Inhibit Pseudomonas aeruginosa at concentration 0.062 mg/ml	11
Aerial	Methanolic	Cup plate method	- Inhibit Streptococcus pneumoniae at concentration 60 mg/ml - Inhibit Proteus vulgaris at concentration 800 mg/ml	33
	Ethanolic		- Inhibit Streptococcus pneumoniae at concentration 60 mg/ml - Inhibit Proteus vulgaris at concentration 60 mg/ml
	Aqueous		- Inhibit Streptococcus pneumoniae at concentration 80 mg/ml - Inhibit Proteus vulgaris at concentration 100 mg/ml
Aerial	Ethanolic	broth microdilution method	Inhibit Staphylococcus aureus at concentration 63 μg/ml	34
Aerial	Methanolic	disk diffusion method	- Inhibit Vibrio cholearea at concentration 400 mg/ml	35

Other species of Euphorbia are known to have anti-bacterial properties as well, such as Euphorbia antiquorum L. and Euphorbia helioscopia L.,, Euphorbia antiquorum L. has anti-bacterial effects against Streptococcus agalactiae, and Euphorbia helioscopia L. has antibacterial effects against Streptococcus aureus, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas multocida, and Klebsiella pneumoniae, (see Table 10).

Table 10

Antibacteria activity of other Euphorbia species.

Species	Part	Methods	Antibacterial activity	Reference
Euphorbia antiquorum L	crude latex	broth microdilution method	Inhibit Streptococcus agalactiae inhibit at concentration 250 μg/ml	36
Euphorbia helioscopia L.	Aerial	disc diffusion method	- Inhibit Escherichia coli - Inhibit Pseudomonas aeruginosa - Inhibit Staphylococcus aureus - Inhibit Pseudomonas multocida - Inhibit Klebsiella pneumoniae	12
Euphorbia helioscopia L	Aerial	disc diffusion method	Inhibit Streptococcus aureus inhibit at concentration 3.9 μg/ml	37
Euphorbia helioscopia L.	Aerial	agar well diffusion method	- Inhibit Escherichia coli - Inhibit Staphylococcus aureus	38
- Euphorbia aleppica L - Euphorbia szovitsii Fisch&Mey. var. harputensis Aznav. ex M. S. Khan - Euphorbia falcata L. sub. falcata var. falcata, - Euphorbia denticulata Lam., - Euphorbia macroclada Boiss., - Euphorbia cheiradenia Boiss.&Hohen, - Euphorbia virgata Waldst.&Kit - Euphorbia petiolata	Aerial	Broth microdilution method	- Inhibit Streptococcus aureus inhibition at 25 μg/ml - Inhibit Streptococcus aureus inhibition at 6.25 μg/ml - Inhibit Streptococcus aureus inhibition at 25 μg/ml - Inhibit Streptococcus aureus inhibition at 25 μg/ml - Inhibit Streptococcus aureus inhibition at 100 μg/ml - Inhibit Streptococcus aureus inhibition at 50 μg/ml - Inhibit Streptococcus aureus inhibition at 12.5 μg/ml - Inhibit Streptococcus aureus inhibition at 25 μg/ml - Inhibit Streptococcus aureus inhibition at 6.25 μg/ml - Inhibit Streptococcus aureus inhibition at 6.25 μg/ml - Inhibit Streptococcus aureus inhibition at 3.12 μg/ml	39

Other Euphorbia species, such as Euphorbia aleppica L., Euphorbia szovitsii, Euphorbia falcata L. sub. falcata var. falcata, Euphorbia denticulata Lam, Euphorbia macroclada Boiss, Euphorbia cheiradenia, Euphorbia virgate, and Euphorbia petiolate, have anti-bacterial effects against Streptococcus aureus. The anti-bacterial properties fight not only Streptococcus species, but also other types of bacteria, such as Pseudomonas aeruginosa, Pseudomonas multocida, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus. (see Table 10).

The three mouthwash formulations used varying concentrations of Euphorbia hirta L.—0.5% in F1; 1% in F2 and 2% in F3. The purpose of this variation is to determine the concentration with the most effective anti-bacterial effects and proper consistency. Manufacturing mouthwash also requires the use of surfactants, such as propylene glycol and aquadest, to help increase solubility and reduce surface tension in oil and water solutions by stabilising the layers formed between the two phases and, thus, produce a clear and stable mouthwash.

In this study, organoleptic, acidity (pH), specific gravity, viscosity, stability, irritation, and contact time tests were conducted on the three mouthwash formulations, and their anti-bacterial effects against Streptococcus mutans were determined. In general, a mouthwash with a concentration of 2% Euphorbia hirta L. had good acidity (pH), specific gravity, viscosity, and stability and tested well in irritation, preference, contact time and anti-bacterial effects against Streptococcus mutans.

The organoleptic test results showed that the three formulations maintained an acceptable and stable smell, taste, and colour during eight weeks of storage. These characteristics were used to determine preference for the formulations. The results showed that F3 was dark brown and had a distinctive smell of oleum menthae piperitae. Acidity (pH) tests aim to ensure stability during storage and avoid irritation to users. If it is too acidic, bacteria can develop easily and cause irritation to the oral mucosa. The results of this analysis showed no significant differences between the pH levels of each formulation of Euphorbia hirta L. mouthwash. The pH levels after storage ranged from 4.0 to 6.5. This increase may be caused by reduced concentrations of Euphorbia hirta L. from evaporation during storage or possibly due to temperature, humidity, or active substances or additives used during preparation.

Freeze-thaw examinations aimed to ensure the stability of mouthwash. This test was conducted on three Euphorbia hirta. L. mouthwash formulations at 4 °C and 40 °C for six cycles. Based on the freeze-thaw stability test results, each of the formulations showed stable preparation properties with no separation. The viscosity of a formulation was particularly important when gargled in the mouth. The viscosity of a formulation being closer to that of water ensured a better experience, and the results showed that formulation viscosity was directly related to weight.

Respondents reported that they preferred the F1 formulation of Euphorbia hirta L. mouthwash, compared with F2 and F3, and rated it 3.7, 3.7, and 3.9. Irritant tests were conducted to determine the irritation levels of Euphorbia hirta L. mouthwash. The highest mucus production was noted in F3 (14.965%), which contained 2% Euphorbia hirta L. extract. These results do not indicate irritation because mucus production was less than 15%. Therefore, this formulation can be assumed to contain only a small number of active substances and, thus, did not cause irritation.

Contact time tests were conducted to determine the time required for mouthwash formulations to inhibit bacteria in the oral cavity, and the best contact time was observed in F3. The anti-bacterial effects of Euphorbia hirta L. mouthwash was tested to determine effectiveness against Streptococcus mutans bacteria. Based on several examinations over an eight-week period, the best formulation is F3, with pH levels ranging from 4.79 to 6.05, the highest viscosity value, and a reduction of bacterial colonies by 57.14%. Higher concentrations of active substances can lead to increased anti-bacterial effects and reduced bacterial colonies. The anti-bacterial effects of Euphorbia hirta L. inhibit the growth of Streptococcus mutans by deactivating certain enzymes in the bacteria, namely, glucosyltransferase (GTF) and fructosyltransferase (FTF). Deactivation of these two enzymes inhibits sucrose from producing glucans and fructose, thus altering the growth of Streptococcus mutans and eliminating the ability to adhere and colonise.

The study limitation is that the results only denote the characteristics and antibacterial activity of Euphorbia hirta L. mouthwash in vitro. However, the study results provide a fundamental basis for understanding the benefits of using Euphorbia hirta L. in a mouthwash. Future research may focus on verifying the benefits and clinical uses of Euphorbia hirta L. mouthwash.

Conclusion

The study results provide proof regarding the use of Euphorbia hirta L. for improving human health. Each of the formulations of Euphorbia hirta L. mouthwash showed no significant differences in either the activity or preference tests. The ethanol extract of Euphorbia hirta L. had ideal physical and stability characteristics for mouthwash and anti-bacterial effects that neutralise Streptococcus mutans. The F3 formulation containing 3% Euphorbia hirta L. showed enhanced characteristics, anti-bacterial effects, and stability, compared with the other two formulations, and the clinical use of this mouthwash must be explored further.

Source of funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

Ethical approval is not required for an in-vitro study. This study was performed by strictly following standard protocols in Pharmaceutical Laboratory, Laboratory of Natural Material Pharmacy, Research and Biopharmaceutical Laboratory, and Sekolah Tinggi Ilmu Farmasi Riau.

Authors contributions

BI designed the study, and supervised, analysed, and interpreted the data, as well as partook in writing the original draft of the article. AL designed the study, and analysed and interpreted data, as well as partook in writing the original draft of the article. SS designed the study, conducted the research, interpreted data, and partook in writing the original draft of the article. UNHA, MDCS, and CKL individually revised the final article. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.