1. INTRODUCTION:

Oral health is key to people general well-being and quality of life.¹Toothpastes undergo tremendous developments to improve the treatment of oral conditions, as caries, bad breath, gingivitis, tartar and dental hypersensitivity. The formulations of toothpastes began in China and India, as 300-500 BC². In recent years, teeth’s whitening is one of the most rapidly growing oral care sectors fueled by consumers’ demand for both healthy and cosmetically attractive smiles.

Nevertheless, access to dental clinics for teeth whitening is restricted to a small part of the population. Therefore, there has been an interest in developing methods so that the patients can apply teeth whitening at home^3,4. Toothpastes are based on optimized abrasive technology to remove and control stains^5,6. Despite the fact that the whitening toothpaste are available and easier-to-use, their whitening effects and impact on teeth have not yet been properly assessed. Whitening dentifrices are composed of different active ingredients, such as chemical agents and high amount of abrasives. Even though the chemical action is not well elucidated, abrasive components associated with tooth brushing seem to be the main factor leading to the teeth whitening effect. However, dentifrices with higher amount of abrasive may produce increased surface roughness in dental tissues⁷. For ages, many cultures have used home remedies composed of natural plants for teeth whitening. These remedies were assumed to be effective, economic and much safer than conventional teeth whitening products. Despite the wide use of these remedies, it is not based on a solid scientific evidence, few scientific reports could be traced^8,9 on their evaluation as teeth whitening agents. Therefore, the aim of our study is to characterize and formulate some of these home remedies and to compare their teeth whitening effect to a conventional teeth whitening toothpaste after tooth brushing at one and six months’ intervals.

2. MATERIALS AND METHODS:

2.1. Materials:

2.1.1. Plant Material:

Salvia officinalis L. (Sage) herb, F. Lamiaceae (250 gm), Fragaria ananassa Duchesne (Strawberry) fruits, F. Rosaceae (2500 gm), Psidium guajava L. (Guava) leaves, F. Myrtaceae (500 gm), Cocos nucifera L. (Coconut) oil, F. Arecaceae (60 ml), Curcuma longa (Turmeric) rhizomes, F. Zingeberaceae (1000 gm) and Citrus limon (Lemon) peels, F. Rutaceae (250 gm) were purchased from the local Egyptian market and Faculty of Agriculture Farm- Cairo University during 2019. With exception to coconut oil, the taxonomical identities of the selected plants were confirmed by the Vegetables and Fruits Department-Faculty of Agriculture- Cairo University. Voucher specimens are kept at the herbarium of faculty of pharmacy, MSA University; (MSA-2019-7, MSA-2019-8, MSA-2019-9, MSA-2019-10 and MSA-2019-11)

2.1.2. Chemicals and reagents:

Sodium lauryl sulfate, sodium bicarbonate, EDTA, camphor, hydroxy propyl methyl cellulose, glycerin was purchased from Sigma Aldrich company and carboxymethyl cellulose (Oxford lab chem, India), all other solvents were of analytical grade. Signal® whitening tooth paste was purchased from local Egyptian market. Folin–Ciocalteu reagent and aluminium chloride were purchased from Sigma Co (St. Louis, MO). Quercetin and gallic acid were obtained from E-merck, Darmstadt, Germany.

2.1.3. Spectrophotometry apparatus for colorimetric study:

UV-visible spectrophotometer, Shimadzu UV (P/N 204-58000) was used to measure the absorbance in UV range.

2.2. Methods:

2.2.1. Preparation of plant extracts:

The plant materials were separately extracted with 70% ethanol until exhaustion. The alcohol is then evaporated using a rotary evaporator at a temperature not exceeding 50^oC. The dried extracts were kept the refrigerator for further chemical and biological investigations.

2.2.2. Colorimetric determination of total phenolic and flavonoids contents of plant extracts:

The phenolic and flavonoid contentswere calculated as gallic acid andquercetin equivalents respectively adopting the method of (Marinova 2005)¹⁰.

2.2.3. High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (HPLC/MS-MS):

HPLC/Ms-Ms analysis was carried out on a XEVO TQD triple quadruple instrument, Waters® corporation, Milford, MA01757 USA, mass spectrometer. The sample (100μg/mL) solution was prepared using HPLC grade solvent of MeOH, filtered using a membrane disc filter (0.2μm) then subjected to LC-ESI-MS analysis. Samples injection volumes (10μL) were injected into the UPLC instrument equipped with reverse phase C-18 column (ACQUITY® UPLC - BEH C18 1.7μm particle size- 2.1 × 50 mm column). Sample mobile phase was prepared by filtering using 0.2μm filter membrane disc and degassed by sonication before injection. Mobile phase elution was made with the flow rate of 0.2mL/min using gradient mobile phase comprising two eluents: eluent A is H₂O acidified with 0.1% formic acid and eluent B is MeOH acidified with 0.1% formic acid. The parameters for analysis were: source temperature 150°C, cone voltage 30 eV, capillary voltage 3 kV, desolvation temperature 440°C, cone gas flow 50 L/h, and desolvation gas flow 900L/h. Mass spectra were detected in the ESI negative ion mode between m/z 100–1000. The peaks and spectra were processed using the Masslynx 4.1® software.

2.2.4. GC Analysis of Fatty acids and Hydrocarbons:

Unsaponifiable matters and fatty acid methyl esters were separated using HP- Hewlett Packard GC-system, series 6890 equipped with Flame Ionization Detector (FID). A capillary column (HP-5 5% phenyl methyl siloxane, 30m x 320μm, film thickness 0.25μm) was used in the separation. The injector port temperature was set 240°C (split mode) and the detector cell at 280°C. The flow rate of the carrier gas, N₂, was 20 ml/min, for H₂ 20 ml/min and for air 200ml/min. The column temperature was 80°C for 1 min and then increased to 280°C by the rate of 8°C/min, with maximum column temperature 325°C then isothermally for 20 minutes.

2.2.5. Preparation of natural toothpastes

Six toothpastes were formulated from the prepared extracts separately by trituration method. A liquid base was prepared first with humectants (glycerin), preservatives (EDTA) and water; to this base herbal extract was added, triturated well with hydroxyl propyl methyl cellulose (HPMC) and kept aside for 15 min then surface active agent was added at the end and mixed slowly and thoroughly to prevent aeration or foaming. The formulation included; 1 gm of each extract (except for coconut oil, where an equivalent of 2mls was added), 0.5gm sodium bicarbonate, 0.4gm sodium lauryl sulphate, 0.7gm hydroxy propyl methyl cellulose, 1 ml glycerin and water (q.s). Mixing was continued till all constituents were evenly distributed. The finished paste was allowed to stand for 24 h, then filled into collapsible tubes, stored and used for further studies¹¹

2.2.6. Evaluation of toothpastes:

Organoleptic parameters:

Organoleptic parameters (colour, odor, texture) were evaluated and visual inspection.

· pH: pH of formulated toothpastes was determined by using pH meter.¹²

· Homogeneity: The toothpaste shall extrude a homogenous mass from the collapsible tube or any suitable container by applying of normal force at 27±20C in addition bulk of contents shall extrude from the crimp of container and then rolled it gradually.¹²

· Foamability: 5g of toothpaste was weighed into a 100ml glass beaker. 10ml of distilled water was added to it and allowed to stand for 30 minutes (this allows the toothpaste to disperse in the water). The contents of the beaker were stirred and the slurry was transferred to a 250ml graduated measuring cylinder. The residue in the beaker was rinsed and transferred with further 5-6ml portion of water to the cylinder. The contents of the cylinder were stirred to ensure a uniform suspension. The cylinder was stoppered and subjected to 12 complete shakes. The cylinder was allowed to stand for 5 minutes and the volume of foam calculated as¹³: Foaming ability = L1-L2; L1 = volume in ml of foam with water L2 = volume in ml of water only.

· Extrudability: The formulated toothpastes were filled in standard capped collapsible aluminum tube and sealed by crimping to the end. The weights of tubes were recorded. The tubes were placed between two glass slides and were clamped. 500g was placed over the slides and then cap was removed. The amount of the extruded paste was collected, weighed and the percentage was calculated.¹⁴

2.2.7. Teeth selection:

Twenty extracted premolars were selected so that they were free of caries and cracks or any damage from extraction, teeth were cleaned from any debris by scaling and stored in distilled water.

2.2.8. Specimens preparation and grouping:

Selected teeth were sectioned at cemento-enamel junction in a mesio-distal direction using a low speed micro-motor and carborundum disc to cut off the roots, and the crown were mounted on acrylic resin mold with labial surface facing upward. The specimens were divided into 4 groups (n=5), as follows:

Group 1: Teeth brushed with Signal White® whitening toothpaste

Group 2: Teeth brushed with Salvia officinalis (Sage) toothpaste (SO)

Group 3: Teeth brushed with Cocos nucifera (Coconut) toothpaste (CN)

Group4: Teeth brushed with Curcuma longa (Turmeric) toothpaste (CL)

2.2.9. Base line color measurement:

Vita easy shade (spectrophotometer) was used to measure the color alteration. Teeth color was analyzed on the basis of color alteration (ΔE), luminosity (∆L), alteration on the green/red axis (Δa) and alteration on the blue/yellow axis (Δb) co-ordinates from CIE lab color system¹⁵. The measurement was performed at base line (T₀) before using any type of toothpaste.

2.2.10. Application of toothpaste:

Toothpastes were applied on the specimens by tooth brushing machine and brushing was done for four minutes and 40 seconds, which was assumed to be equivalent to 1 month brushing, followed by color measurement using spectrophotometer. Then continuing brushing for 24 minutes which is equivalent to 5 months brushing, thus, the brushing time would be equivalent to brushing 6 months¹⁶. It was done with 4 minutes and 40 seconds brushing in the first day, followed by 4 minutes brushing for 5 days and specimens were stored in distilled water between brushing. Teeth’s brushing was carried out using tooth brushing machine which was standardized at a speed of 365rpm, while loading was standardized at 200gm. The tooth brush used has rounded end, uniform length, flexibility and medium bristles.

2.2.11. Color measurement after 1 and 6 months’ intervals:

Color measurement was taken after brushing for the equivalent of 1 month (T₁) and 6 months (T₂). In each specimen, measurements were the mean of triplicates at different points.

2.3. Statistical analysis:

The mean and standard deviation values were calculated for each group in each test. Data were explored for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests, data showed non-parametric distribution. Kruskal Wallis test was used to compare between more than two groups in non-related samples. Mann Whitney was used to compare between two groups in non-related samples. Wilcox on test was used to compare between two groups in related samples. The significance level was set at P ≤ 0.05. Statistical analysis was performed with IBM® SPSS® Statistics Version 20.

3. RESULTS:

3.1. Colorimetric determination of total phenolic and flavonoids contents of plant extracts:

S. officinalis extract showed the highest phenolic content followed by P. guajava extract and C. longa extract. As for the flavonoids content, C. longa extractshowed the highest concentration. On the other hand, C. limon and F. ananassa extracts have the lowest phenolic and flavonoids contents. (Table 1)

3.2. HPLC/MS-MS analyses of plant extracts:

As shown in (Table 2), S. officinalis, P. guajava, C. longa, C. limon, F. ananassa extracts were profiled via HPLC/MS-MS fingerprints aiming at tentatively identifying their metabolites. The peaks and spectra were processed using the (Masslynx® 4.1) software and identified by comparing its retention time and mass fragmentation patterns with data reported in the literature. A total of sixty-five metabolites were identified belonging to different classes viz flavonoids, anthocyanins, tannins, terpenoids, phenolic compounds and acids derivatives. twenty-six in the hydro-alcoholic extract of C. longa rhizomes, while nine were identified in each of C. limon fruit peels and S. officinalis herb extract. 10 were identified in P. guajava leaves extract and eleven in F. ananassa fruits extract.

Table 1: Total Phenolic and flavonoids content of the extracts with Folin–Ciocalteu at λmax 750 nm and 0.1 M AlCl₃ at λmax 420 nm

Extract	Total Phenolic content Concentration (mg/g)	Total Flavonoids content Concentration (mg/g)
P. guajava	102.73	26.14
F. ananassa	2.35	1.70
C. longa	102.36	93.39
C. limon	51.6	5.75
S. officinalis	196.49	16.87

Table 2: Results of HPLC-MS/MS analysis of C. longa, C. limon, S. officinalis, P. guajava and F. ananassa extracts

Peak No.	Retention time	Molecular weight	[M-H]^-	Base Peak/ Fragments	Molecular Formula	Identification	Reference
*C. longa*
1	1.43	170	169	125, 107, 97, 79	C₇H₆O₅	Gallic acid	27
2	1.70	198	197	153	C₉H₁₀O₅	Syringic acid	28
3	2.36	154	153	153, 109	C₇H₆O₄	Protochatecuic acid	28
4	3.14	344	343	181	C₉H₁₀O₄	Homovanillic acid-O-hexoside	28
5	3.58	354	353	191,179	C₁₆H₁₇O₉	Chlorogenic acid	29
6	4.84	180	179	161, 135	C₉H₈O₄	Caffeic acid	27
7	5.78	356	355	193, 179	C₁₆H₂₀O₉	Ferulic acid-O-hexoside	28
8	5.98	154	153	42	C₁₀H₁₈O	Eucalyptol	30
9	6.81	136	135	135	C₁₀H₁₆	Terpinoline	20
10	7.03	168	167	152, 124	C₈H₈O₄	Vanillic acid	28
11	7.90	164	163	147	C₉H₈O₃	Coumaric acid	28
12	8.22	194	193	193, 179	C₁₀H₁₀O₄	Ferulic acid	28
13	8.68	610	609	301, 447	C₂₇H₃₀O₁₆	Rutin	31
14	9.08	464	463	301, 271, 255	C₂₁H₁₉O₁₂	Quercetin-3-O-glucoside	29
15	9.15	448	447	285, 255	C₂₁H₂₀O₁₁	Kaempferol-3-O-glucoside	29
16	10.91	610	609	463,301	C₂₈H₃₄O₁₅	Hesperidin	27
17	12.05	360	359	197, 161	C₁₈H₁₆O₈	Rosmarinic acid	28
18	12.19	434	433	271, 177, 151	C₂₇H₃₂O₁₄	Naringin-O-hexoside	28
19	14.78	204	203	93	C₁₅H₂₄	β-Caryophylline	30
20	15.33	302	301	271, 255	C₁₅H₁₀O₇	Quercetin	29
21	16.41	202	201	131, 118	C₁₅H₂₂	α-Curcumene	30
22	17.40	272	271	177, 151	C₁₅H₁₂O₅	Naringenin	31
23	17.55	270	269	269	C₁₅H₁₀O₅	Apigenin	27
24	17.91	302	301	258, 143	C₁₆H₁₄O₆	Hesperitin	28
25	18.83	368	367	271	C₂₁H₂₀O₆	Curcumin	32
26	21.6 1	218	217	121	C₁₅H₂₀O	Turmerone	30
*C. limon*
1	4.21	740	739	269	C₂₄H₂₃O₁₄	Apigenin 7-O-neohesperidoside-6-C-glucoside	33
2	9.01	772	771	463, 301, 271, 255	C₃₃H₄₀O₂₁	Quercetin 7-O-glucoside-3-O-rutinoside	33
3	10.11	640	639	315, 271	C₂₂H₂₂O₁₂	Isorhamnetin-3-O-di-glucoside	33
4	12.12	624	623	299	C₂₇H₃₀O₁₆	Diosmetin 6,8-di-C-glucoside	33
5	12.23	608	607	301	C₂₈H₃₂O₁₅	Neodiosmin	33
6	17.51	578	577	401, 293, 269	C₂₇H₃₀O₁₄	Rhoifolin	31
7	19.12	598	597	597	C₂₇H₃₂O₁₅	Eriocitrin	33
8	31.23	300	299	241	C₁₆H₁₂O₆	Diosmetin	33
9	31.53	594	593	285	C₂₇H₃₀O₁₅	Luteolin-O-rutinoside	34
*S. officinalis*
1	11.72	446	445	385, 283	C₂₇H₃₀O₁₅	Acacetin hexoside	35
2	13.21	464	463	301, 271, 255	C₂₁H₁₉O₁₂	Quercetin-3-O-glucoside	29
3	15.31	448	447	285, 255	C₂₁H₂₀O₁₁	Kaempferol-3-O-glucoside	29
4	16.31	448	447	285	C₂₁H₂₀O₁₁	Luteolin-3-O-glucoside	36
5	16.65	316	315	271, 287, 285	C₁₆H₁₂O₇	Isorhamnetin	39
6	17.72	610	609	463,301	C₂₈H₃₄O₁₅	Hesperidin	37
7	19.73	288	287	271, 285, 223	C₁₅H₁₂O₆	Eriodictyol	36
8	21.12	300	299	299	C₁₇H₁₄O₅	4-Hydroxy-5,7- dimethoxyflavanone	37
9	24.32	270	269	269	C₁₅H₁₀O₅	Apigenin	27
*P. guajava*
1	5.91	170	169	125, 107, 97, 79	C₇H₆O₅	Gallic acid	27
2	6.54	290	289	271,245	C₁₅H₁₄O₆	Catechin	31
3	7.74	306	305	271, 191	C₁₅H₁₄O₇	Gallocatechin	29
4	8.53	578	577	451, 425, 407, 289	C₃₀H₂₆O₁₂	Procyanidin B1	31
5	12.51	464	463	301, 271, 255	C₂₁H₂₀O₁₂	Quercetin-3-O-glucoside	19
6	24.46	272	271	177, 151	C₁₅H₁₂O₅	Naringenin	31
7	30.81	464	463	301, 271, 255	C₂₁H₂₀O₁₂	Quercetin-3-O-galactoside	38
8	33.71	434	433	301, 271, 255	C₂₀H₁₈O₁₁	Quercetin-3-O-arabinoside	38
9	46.52	318	317	273, 258	C₁₅H₁₀O₈	Myricetin	39
*F. ananassa*
1	2.52	286	285	255, 241, 229, 213	C₁₅H₁₀O₆	Kaempferol	29
2	3.03	382	381.1	191,111	C₇H₁₂O₆	Quinic acid derivative	40
3	9.81	866	865	577	C₄₅H₃₈O₁₈	Proanthocyanidin trimer	36
4	18.52	595	595	270	C₂₇H₃₁O₁₆	Pelargonidin-3-O-diglucoside	31
5	19.26	580	579.2	271, 235, 177, 151	C₂₇H₃₂O₁₄	Naringin	31
6	20.10	290	289	271,245	C₁₅H₁₄O₆	Catechin	31
7	27.14	580	579	270	C₂₇H₃₁O₁₄	Pelargonidin-3-O-rutinoside	31
8	31.80	302	301	257, 229, 185	C₁₄H₇O₈	Ellagic acid	32
9	32.22	478	477	301	C₂₁H₁₈O₁₃	Quercetin-3-O-glucuronide	34
10	36.01	534	533	285, 255	C₃₀H₃₂O₁₉	Kaempferol-malonyl-hexoside	34
11	42.80	594	593	163	C₃₀H₂₇O₁₃	Coumaroyl glucoside	23

3.3. GLC analysis of the identified fatty acids and unsaponifiable matters of C. nucifera oil:

C. nucifera oil consists of medium chain triglycerides.¹⁷ A total of twenty unsaponfiable hydrocarbons were identified inC. nuciferaoil by comparing the relative retention time of the peaks with those of the authentic compounds. The major of which was n- Triacontane. Concerning the saturated fatty acids, six compounds were identified, where; lauric acid represented (53.11%) and myristic acid represented (20.42%). Only two unsaturated fatty acids were identified in the oil in a relatively low percentage, which are oleic acid (3.95%) and linoleic acid (0.46%). (Table 3).

Table 3: Results of GLC analysis of the identified fatty acids and unsaponifiable matters of C. nucifera oil

Compound	Retention time (min.)	Relative %
Unsaponifiable matters
C14 n-Tetradecane	11.31	0.09
C15 Pentadecane	12.71	0.27
C16 n-Hexadecane	14.01	0.76
C17 n-Heptadecane	15.33	2.56
C18 n-Octadecane	15.99	2.94
C19 n-Nonadecane	17.81	3.78
C21 n-Henicosane	20.01	7.31
C22 n-Docosane	21.11	7.86
C23 n-Tricosane	22.31	9.02
C24 n-Tetracosane	22.92	7.88
C25 n-Pentacosane	24.00	3.28
C26 n-Hexacosane	24.71	3.05
C27 n-Heptacosane	26.21	8.59
C28 n-Octacosane	26.52	1.99
C29 n-Nonacosane	27.23	3.40
C30 n-Triacontane	28.64	17.10
Cholesterol	ND*	ND*
Campasterol	31.51	2.45
Stigmasterol	32.90	0.45
Alpha –amyrine	33.79	0.49
Total identified %		83.27%
Saturated fatty acids
Caprylic acid C8	6.0	7.52
Capric acid C10	7.4	5.93
Lauric acid C12	9.5	53.11
Myristic acid C14	12.0	20.42
Palmitic acid C16	14.9	7.13
Stearic acid C18	18.1	1.48
Unsaturated fatty acids
Oleic acid C18(1)	18.5	3.95
Linoleic acid C18(2)	19.6	0.46
Total identified %		100%

*ND: Not detected

3.4. Physical examination of toothpastes:

The formulated natural toothpastes were of different colors and showed the good homogeneity with absence of lumps. The toothpastes were also, smooth in nature, foamability around 10, pH 5 to 8.2, and extrudability 95%. The physical characters of the formulated toothpastes are presented in (Table 4).

Teeth whitening evaluation was applied only on the best formulated natural toothpastes based on their good stability and foamability characters and thus, C.nucifera toothpaste (CN), C.longa toothpaste (CL) and S.officinalis toothpaste (SO) were further investigated. Photos of the best formulated extracts were shown in (Figure 1).

4. DISCUSSION:

Conventional teeth whitening toothpastes are composed of chemicals that cause undesirable side effects for instance; allergies, irritation and mucosal ulceration. In addition, the application of teeth bleaching agents as hydrogen peroxide, or its derivative; car bamide peroxide is potentially carcinogenic due to free radicals produced by them. Thus, there is public perception that herbal-based home remedies provide for more economic and safer options, which account for the increased preference for natural toothpastes.⁸ In the present study, we characterized the metabolomic profiles of selected plants that were used in some home remedies for teeth whitening. Hydro-ethanolic extracts of S.officinalis, C.longa, F.ananassa, C.limonand P.guajavawere evaluated for their phenolic and flavonoid contents and S. officinalis and C.longaextracts showed the highest phenolic andflavonoids contents respectively. HPLC/MS-MS analyses revealed the presence of sixty-five belonging to different classes viz flavonoids, anthocyanins, tannins, terpenoids, phenolic compounds and acids derivatives. The majority of the compounds are phenolic and polyphenolic which are well-known for their acidic character.¹⁸ The whitening action of these extracts may be attributed to this acidic character as it causes subsequent acid erosion and demineralization in a similar mechanism to that of peroxides. Moreover, this acidic character promotes their antimicrobial property which is considered an add-on in their employment in oral hygiene formulations. To evaluate the formulated toothpastes, we observed the color changes in dental enamel surface after brushing with different tooth pastes at (T₀), (T₁) and (T₂). There was no significant difference in the DL, Db and Da. Nagpal and Sood (2013)¹⁹ stated that the 100% virgin coconut oil had a broad-spectrum antibacterial effect which has the same action as chlorohexidine. In our study, coconut toothpaste showed no significant difference in color changes DL, Db and Da between T₀ and T₁ and between T₀ and T₂, also, concerning DE, it showed no changes that can be seen by the observers clinically where DE<1. On the other hand, turmeric has anti-microbial effect, its use in oral health as periodontal disease was investigated by Pisani et al.2010¹⁶ and they reported that it can be used as pit and fissure sealant, mouth wash and sub gingival irrigate. In our study, turmeric toothpaste showed no significant difference in DL, Db and Da when compared the color changes at T₀, T₁ and T₂, while regarding D E, it showed changes that can be easily observed where D E >3.3. Therefore, the turmeric can be used as mouth wash, but according to our study not recommended to be used in toothpastes, as it induced color change which was observed toward the dark direction. It has been reported that sage exerts a range of therapeutic activities including anti-bacterial effects²⁰. By evaluating the enamel color changes, it was found that there was no significant difference in DL, Db and Da, but D E showed changes in which D E > 3.3 which can be easily observed clinically. As for Signal white®, there was change in color where D E >3.3 with lighter effect at (T₁), while at (T₂) became slight darker but still lighter than (T₀). Our findings come in accordance with, an in vitro study with a duration of 14 days compared the effect of a chemical teeth whitening toothpaste and teeth whitening toothpaste containing herbal ingredients on human enamel and their findings proved that the chemical whitening toothpaste was more effective, however, it caused increased surface irregularities on the surface of the enamel compared to the herbal one⁸. In addition, Abidia et al. 2019⁹ compared the effect of turmeric and strawberries, among other preparations regarding the enamel shade at 5 and 10 days’ intervals and their results showed that ∆E showed a significant difference in all groups except the turmeric group. Also, Gautam et al. 2020²¹ used aloe vera gel, clove oil, neem powder, pomegranate peel powder and trikatu in herbal toothpaste and proved that it is as good as the marketed herbal formulations. Finally, it is recommended that the formulated toothpastes of sage and coconut can be employed as teeth whitening preparations mainly for their anti-bacterial effect, while the turmeric toothpaste increased the teeth yellowness and thus should be avoided in teeth whitening preparations however it can be used as mouth wash or gel application on the gingiva.

5. CONCLUSION:

Commercial whitening toothpaste increased the lightness of the dental enamel surface after one month, but after six months it did not give the same effect. Sage toothpaste changed the color of enamel with results comparable to those of the conventional toothpaste.

6. CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

7. AUTHORS CONTRIBUTIONS:

Dr. El- Bishbishy suggested the research point, prepared the extracts and performed the phytochemical analyses. Dr. Mostafa formulated and evaluated the physical characters of toothpastes. Dr Hamza and Dr Taher equally contributed in performing the dental studies and statistics. Dr. El-Bishbishy drafted the manuscript. All authors read and approved the final manuscript.

8. REFERENCES:

1. Prasanth M, Ratha S N. Antimicrobial Property of Herbal Toothpastes: An In-Vitro Analysis. Research Journal of Pharmacology and Pharmacodynamics. 2014; 6(1): 30-35

2. Jagtap AM, Kaulage SR, Kanse SS, Shelke VD, Gavade AS, Vambhurkar GB, Todkar RR, Dange VN. Preparation and Evaluation of Toothpaste. Asian J. Pharm. Ana. 2018; 8(4): 191-194.

3. Claydon N, Moran J, Bosma ML, Shirodaria S, Addy M, Newcombe R. Clinical study to compare the effectiveness of a test whitening toothpaste with a commercial whitening toothpaste at inhibiting dental stain. Journal of Clinical Periodontology. 2004;31:1088-1091.

4. Pintado-Palomino K, Vasconcelos CV, Silva RJ, et al. Effect of whitening dentifrices: a double-blind randomized controlled trial. Brazilian Oral Research. 2016; 30(1): e82.

5. Joiner A, Hopkinson I, Deng Y, Westland S. A review of tooth colour and whiteness. Journal of Dentistry. 2008; 36 Suppl 1:S2-7.

6. Khan MK, Bokhari SA, Haleem A, et al. Extrinsic stain removal with a toothpowder: A randomized controlled trial. International Journal of Health Sciences. 2014; 8(3): 269-274.

7. De Menezes M, Turssi CP, Hara AT, Messias DC, Serra MC. Abrasion of eroded root dentine brushed with different toothpastes. Clinical Oral Investigations. 2004; 8(3): 151-155.

8. Kalliath C, Mukunda A, Pynadath M, Venugopal V, Prethweeraj J. Comparison between the effect of commercially available chemical teeth whitening paste and teeth whitening paste containing ingredients of herbal origin on human enamel. Ayu. 2018;39(2):113-117.

9. Abidia R, El-Hejazi AA, Azam A, Al-Qhatani S, Al-Mugbel K, AlSulami M. Efficacy of Tooth Whitening Using Natural Products in Vitro. The Saudi Dental Journal. 2019; 31: S26.

10. Marinova. Total phenolics and total flavonoids in bulgarian fruits and vegetables. Journal of the University of Chemical Technology and Metallurgy. 2005; 40(3): 255-260.

11. Sharma P. Cosmetics- Formulation, manufacturing and quality control. 4th ed: Vandana Publishing House; 2010.

12. George D, Bhat SS, Antony B. Comparative evaluation of the antimicrobial efficacy of aloe vera tooth gel and two popular commercial toothpastes: an in vitro study. General dentistry. 2009; 57(3): 238-241.

13. Singh K, Singh P, Oberoi G. Comparative studies between herbal toothpaste (dantkanti) and non-herbal tooth paste. 2016; 4: 53-56.

14. Solomon CU. Preliminary phytochemical screening of different solvent extracts of stem bark and roots of Dennettia tripetala G. Baker. Asian Journal of Plant Science and Research. 2013; 3(3): 10-13.

15. White DJ. Development of an improved whitening dentifrice based upon "stain-specific soft silica" technology. The Journal of Clinical Dentistry. 2001; 12(2): 25-29.

16. Pisani MX, Bruhn JP, Paranhos HF, Silva-Lovato CH, de Souza RF, Panzeri H. Evaluation of the abrasiveness of dentifrices for complete dentures. Journal of prosthodontics : Official Journal of the American College of Prosthodontists. 2010; 19(5): 369-373.

17. Tripathi S, Sahu Uk, Meshram J, Kumari R, Tripathi DK, Ajazuddin, Alexander A, Sharma H, Sahu GK. Formulation and characterization of Virgin Coconut Oil Emulsion (VCOE) for treatment of Alzheimer’s disease. Res. J. Pharma. Dosage Forms and Tech. 2018; 10(2): 49-54.

18. Pandian RS, Noora AT. GC-MS Analysis of Phytochemical Compounds Present in the Leaves of Citrus medica. L. Research J. Pharm. and Tech. 2019; 12(4): 1823-1826.

19. Nagpal M, Sood S.Role of curcumin in systemic and oral health:An overview. J Nat Sci Biol Med. 2013; 4(1): 3-7.

20. Beheshti-Rouy M, Azarsina M, Rezaie-Soufi L, Alikhani MY, Roshanaie G, Komaki S. The antibacterial effect of sage extract (Salvia officinalis) mouthwash against Streptococcus mutans in dental plaque: a randomized clinical trial. Iranian Journal of Microbiology. 2015; 7(3): 173-177.

21. Gautam D, Palkar P, Maule K, Singh S, Sawant G, Kuvalekar C, Rukari T, Jagtap VA. Preparation,Evaluation and Comparison of Herbal toothpaste with marketed Herbal toothpaste. Asian J. Pharm. Tech. 2020; 10(3): 165-169

22. Waly NE, Aborehab NM, Bishbishy MHE. Chemical fingerprint of Bacopa monnieri L. and Rosmarinus officinalis L. and their neuroprotective activity against Alzheimers disease in rat models putative modulation via cholinergic and monoaminergic pathways. Journal of Medicinal Plants Research. 2019; 13: 252-268.

23. Vallverdú-Queralt A, Regueiro J, Alvarenga JFR, Martinez-Huelamo M, Leal LN, Lamuela-Raventos RM. Characterization of the phenolic and antioxidant profiles of selected culinary herbs and spices: caraway, turmeric, dill, marjoram and nutmeg. Food Science and Technology. 2015; 35: 189-195.

24. Bakr RO, El Bishbishy MH. Profile of bioactive compounds of Capparis spinosa var. aegyptiaca growing in Egypt. Revista Brasileira de Farmacognosia. 2016; 26(4): 514-520.

25. Arivukkarasu R, Rajasekaran A, bin hussain Sh, Ajnas M.Simultaneous detection of Rutin, Quercetin, Gallic acid, Caffeic acid, Ferulic acid, Coumarin, Mangiferin and Catechin in Hepatoprotective commercial herbal formulations by HPTLC technique. Res. J. Pharmacognosy and Phytochem. 2018; 10(1): 59-62.

26. Lee J, Jung Y, Shin J-H, et al. Secondary metabolite profiling of Curcuma species grown at different locations using GC/TOF and UPLC/Q-TOF MS. Molecules (Basel, Switzerland). 2014; 19(7): 9535-9551.

27. Elhawary SS, Younis IY, El Bishbishy MH, Khattab AR. LC–MS/MS-based chemometric analysis of phytochemical diversity in 13 Ficus spp. (Moraceae): Correlation to their in vitro antimicrobial and in silico quorum sensing inhibitory activities. Industrial Crops and Products. 2018; 126: 261-271.

28. 28.Isrul M, Juliansyah R, Saleh A, Yuliastri WO, Pusmarani JH, Maulidina WOW. Phytochemical Analysis, Standardization and Cytotoxic Activity of Curcuma aureginosa Extract in Human Breast Cancer (MCF-7) Cell Line.Research J. Pharm. and Tech. 2019; 12(4): 1967-1973

29. Aborehab NM, El Bishbishy MH, Refaiy A, Waly NE. A putative Chondroprotective role for IL-1β and MPO in herbal treatment of experimental osteoarthritis. BMC Complement Altern Med. 2017; 17(1): 495.

30. Brito A, Ramirez JE, Areche C, Sepúlveda B, Simirgiotis MJ. HPLC-UV-MS profiles of phenolic compounds and antioxidant activity of fruits from three citrus species consumed in Northern Chile. Molecules (Basel, Switzerland). 2014; 19(11): 17400-17421.

31. Ezzat SM, El Bishbishy MH, Aborehab NM, et al. Upregulation of MC4R and PPAR-α expression mediates the anti-obesity activity of Moringa oleifera Lam. in high-fat diet-induced obesity in rats. Journal of Ethnopharmacology. 2020; 251: 112541.

32. Wojakowska A, Piasecka A, García-López PM, et al. Structural analysis and profiling of phenolic secondary metabolites of Mexican lupine species using LC–MS techniques. Phytochemistry. 2013; 92: 71-86.

33. Ibrahim RM, El-Halawany AM, Saleh DO, El Naggar EMB, El-Shabrawy AE-RO, El-Hawary SS. HPLC-DAD-MS/MS profiling of phenolics from Securigera securidaca flowers and its anti-hyperglycemic and anti-hyperlipidemic activities. Revista Brasileira de Farmacognosia. 2015; 25: 134-141.

34. Wang Y, Yang L, He Y-Q, et al. Characterization of fifty-one flavonoids in a Chinese herbal prescription Longdan Xiegan Decoction by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and photodiode array detection. Rapid Communications in Mass Spectrometry. 2008; 22(12): 1767-1778.

35. Subramanian G, Meyyanathan SN, Gowramma B, Palanisamy DS. Liquid chromatography-mass spectrometric method for simultaneous estimation of apigenin and luteolin from Achillea millefolium Linn. Asian J. Research Chem. 2016; 9(12): 629-632.

36. Simirgiotis MJ, Benites J, Areche C, Sepúlveda B. Antioxidant Capacities and Analysis of Phenolic Compounds in Three Endemic Nolana Species by HPLC-PDA-ESI-MS. Molecules (Basel, Switzerland). 2015; 20(6): 11490-11507.

37. Kajdzanoska M, Petreska J, Stefova M. Comparison of different extraction solvent mixtures for characterization of phenolic compounds in strawberries. J Agric Food Chem. 2011; 59(10): 5272-5278.

38. Prakash O, Mahapatra DK, Singh R, Singh N, Verma N, Akash V. Development of a New Isolation Technique and Validated RP-HPLCmethod for Quercetin and Kaempferol from Azadirachta indica leaves. Asian J. Pharm. Ana. 2018; 8(3): 164-168.

39. El Bishbishy MH, Gad HA, Aborehab NM. Chemometric discrimination of three Pistacia species via their metabolic profiling and their possible in vitro effects on memory functions. Journal of Pharmaceutical and Biomedical Analysis. 2020; 177: 112840.

40. Mullen W, Yokota T, Lean ME, Crozier A. Analysis of ellagitannins and conjugates of ellagic acid and quercetin in raspberry fruits by LC-MSn. Phytochemistry. 2003; 64(2): 617-624.

41. Singh RK, Chang HW, Yan D, et al. Influence of diet on the gut microbiome and implications for human health. Journal of Translational Medicine. 2017; 15(1): 73.

42. Vardhan MS, Krishna KVVS, Sankar KG, Krishna SR, Mani TT. Simultaneous Estimation of Ellagic acid and Quercetin in Marketed Ayurvedic Formulations by HPTLC.Res. J. Pharmacognosy and Phytochem. 2017; 9(4): 223-227

Received on 21.08.2020 Modified on 09.09.2020

Research J. Pharm. and Tech. 2021; 14(7):3639-3646.

DOI: 10.52711/0974-360X.2021.00629

	*C. longa* toothpaste	*S. officinalis* toothpaste	*C. nucifera* toothpaste	*P. guajava* toothpaste	*F. ananassa* toothpaste	*C. limon* toothpaste
Color	Reddish brown	Brown	Creamy	Brown	Red	Yellow
pH	7.5	8	8.5	7	4.5	5
Homogeneity	good	very good	Good	soft	Soft	Soft
Foamability	11(good)	10(good)	12(good)	6 (bad)	7(bad)	10 (good)
Extrudability	95%	90%	85%	63 %	50 %	60%

Tooth-paste	Change in the color of enamel (∆E)		Lightness of enamel (∆L)		Opponent dimensions on a red/green axis (∆a)		Opponent dimensions on a yellow/blue axis (∆b)
Tooth-paste	T₀ and T₁ intervals	T₀ and T₂ intervals	T₀ and T₁ intervals	T₀ and T₂ intervals	T₀ and T₁ intervals	T₀ and T₂ intervals	T₀ and T₁ intervals	T₀ and T₂ intervals
Signal White®	17.82 ± 1.31	13.15 ± 0.99	13.00 ± 1.22	7.60 ± 0.74	-2.88 ± 1.03	-2.18 ± 0.46	-12.38 ± 0.98	-9.73 ± 0.52
SO	3.39 ± 1.15	8.05 ± 2.41	-3.33 ± 1.10	0.77 ± 5.77	-0.20 ± 0.35	-2.08 ± 1.88	-0.23 ± 0.65	-3.20 ± 5.92
CL	5.67 ± 1.54	5.02 ± 2.08	-0.08 ± 1.81	-2.90 ± 2.96	0.33 ± 1.14	-0.23 ± 0.40	5.40 ± 1.69	2.50 ± 4.75
CN	1.25 ± 0.77	6.18 ± 5.32	0.53 ± 0.40	-5.80 ± 5.34	-0.23 ±0.42	0.25± 1.55	-0.13 ± 0.31	0.68 ± 1.64