Identification of Medicinal Chemicals in Herbal Medicine for Shortness of Breath in Cirebon Using Thin-Layer Chromatography Method

. Herbal medicine is prohibited from adding Medicinal Chemicals (BKO), both isolated and synthetic, by Permenkes No. 007 of 2012 Article 7. However, several previous studies found herbal medicine containing BKO; the addition of BKO is indicated so that products from the manufacturer feel faster and have instant efficacy. However, the addition of BKO to herbal medicine has side effects on the organs of the consumer. In 2014 – 2022, BPOM released 12 herbal products that were proven to contain BKO. This study aims to identify the existence of BKO in shortness-of-breath herbal medicine and to find out what BKO is contained in the herbal medicine. Using the purposive sampling method, this study tested six herbal medicine samples with shortness of breath circulating in Cirebon. The techniques used in the identification were thin-layer chromatography (KLT) with the silent phase of GF 254 silica gel, the motion phase of Chloroform, Methanol, and Aquabidest with a ratio of 2: 7: 1. Spot detection through 254 nm UV light. The controlled solution is the test solution (A), the positive control solution (B), and the comparator raw solution (C). Based on the study's results, 5 out of 6 herbal samples containing BKO, Samples 1 and 3 contained CTM and Ephedrine Hcl. Samples 2, 5, and 6 contained Ephedrine HCl only. Sample 4 does not contain BKO.


INTRODUCTION
Shortness of breath is one of the symptoms of respiratory tract disorders.The prevalence of respiratory diseases in Cirebon City and Regency is relatively high.
Referring to West Java RISKESDAS data in 2018, the prevalence of Acute Respiratory Infections in Cirebon Regency was 2.79%, and in Cirebon City was 3.65%.The prevalence of Pulmonary Tuberculosis in Cirebon Regency is 0.37%, and in Cirebon City is 0.35%.
The prevalence of asthma diagnosis in Cirebon Regency is 1.03%, and in Cirebon City is 1.27%.The prevalence of asthma recurrence in Cirebon Regency is 56.66%, and in Cirebon City, it is 61.53% (Ministry of Health RI, 2019) sarian (galenik), or a mixture of these ingredients has been used for treatment for generations, and can be applied following the norms that apply in society (regulation the Minister of Health, 2012).In line with the opinion of Parwata, who states that Traditional Medicine is an ingredient or ingredient in the form of plant materials, animal materials, mineral ingredients, galenic preparations or mixtures, and these ingredients, which have traditionally been used for treatment based on experience (Parwata, 2016).Indonesian society is very thick with traditional nuances; this was expressed by the POM Agency, which stated that this tendency to return to nature is based on the general reason that natural medicines are safe to use and easy to obtain (BPOM, 2018).However, with this tendency, there is a negative effect, namely a violation of regulations on the herbal medicine production process, namely the mixing of medicinal chemicals (BKO) in herbal preparations.He referred to the Indonesian Minister of Health Regulation No. 007 of 2012, which states that "medicinal chemicals are prohibited from being added to herbal medicine" (Minister of Health, 2019).This has made various parties interested in researching the existence of BKO in Jammu's shortness of breath.Shortness of breath herbal medicine in Malang contained theophylline.Likewise, the results of Herdini's research stated that his research on three samples of herbal medicine for shortness of breath in Bekasi City that were tested, three samples of herbal medicine were positive for theophylline (Hardini et al., 2021).The latest research related to BKO in shortness-of-breath herbs was carried out by Aziza, stating that of the six samples of shortness-of-breath herbs circulating in the city of Banjarmasin, all positive samples contained theophylline (Aziza, 2022).Findings related to illegal herbal medicine can be seen in the results of BPOM testing from November 2014 to October 2022 and various studies on the existence of BKO in herbal medicine.One published article states that chlorpheniramine maleate is contained in 9 traditional medicine preparations, and Ephedrine HCl is contained in 3 traditional medicine preparations (BPOM, 2018).
Chlorpheniramine maleate (CTM) is one of the drugs that belong to the class of antihistamines that work to minimize inflammatory reactions and is the most widely used antihistamine (Saputra et al., 2018).Ephedrine Hydrochloride is a decongestant drug, namely an adrenergic α-1 receptor stimulant (Kurniawati et al., 2023).Ephedrine HCl can be an adrenergic agent agonist, antiasthma, and bronchodilator (Nurcahyanti, 2018).

METHODS
The research conducted is a type of descriptive research, namely by identifying the drug chemicals Chlorpheniramine Maleate and Ephedrine HCl in herbal medicine for

Organoleptic and Packaging Observations
The research began by observing the packaging, including the registration number, composition, type of packaging, production address, and expiration date of the herbal medicine packaging.Meanwhile, organoleptic observations include the shape, color, odor, and homogeneity of the clock preparation (Utami et al., 2019).

Preparation of Test Solution A (sample)
Each sample of herbs as a fine powder is put into 150 ml of Erlenmeyer, 50 ml of chloroform-methanol mixture (4:1), and a homogeneous shake is added.The filtrate is evaporated in a bucket of water at approximately 70oC until dry.The remaining evaporation is dissolved in 8 ml of methanol and then filtered.Put in vial (Wulandari et al., 2013).

Preparation of Bc Solution (positive control of Chlorpheniramine Marate)
Each sample of herbs as a fine powder is put into 150 ml of Erlenmeyer.Add chlorpheniramine maleate tablet powder equivalent to 12 mg, as many as three tablets with a dose strength of 4 mg each, add 50 ml of chloroform-methanol mixture (4:1), and shake homogeneously.The filtrate is evaporated in a bucket of water at a temperature of approximately 70 o C until dry.The remaining evaporation is dissolved in 8 methanol and then filtered.Put in vial (Wulandari et al., 2013).

Preparation of Be Solution (positive control of Ephedrine HCl)
Make an injection dilution of Ephedrine HCl 50mg/ml ad 5ml with aqua pro injection.The dilution result is equivalent to 10mg/ml.Each sample of herbal medicine as a fine powder is put into 150 ml of Erlenmeyer, 50 ml of chloroform-methanol mixture (4:1) is added, 1 ml of Ephedrine HCl injection dilution is added, and a homogeneous shake is added.The filtrate is evaporated on a bucket of water at a temperature of approximately 70 o C until dry.The remaining evaporation is dissolved in 8 ml of methanol, then filtered.Put in vial (Wulandari et al., 2013).

Preparation of Cc Solution (Chlorpheniramin Maleate Comparative Standard)
A raw solution of chlorpheniramin maleate was made with 12 mg of chlorpheniramin maleate, then dissolved in 20 ml of methanol.Strain and place in vials (Panjaitan, 1999).

Preparation of Ce Solution (Ephedrine HCl Comparative Standard)
Ephedrine HCl raw solution is made by concentrating 1 ml of Ephedrine HCl injection 50mg/ml in a dish over a water bath until dry, then diluted with 1 ml of methanol.
Put it into the vial.

Motion Phase Orientation
Cut the GF 254 silica gel plate into 3 x 10 cm plates, then activate the plates at 110oC in the oven for 30 minutes.Make a line of 1.5 cm from the bottom, then a creep distance of 8 cm, and a distance of about 0.5 cm from the top border.Put the filter paper into the chamber, then enter the motion phase of Chloroform -Methanol -Aquabidest in a ratio of 2:7:1, and leave it until saturated.Apply two dotting marks to the line on the plate's bottom.
Point the comparison solution (Ce and Cc) on the plate.The plate is inserted into the saturated chamber, closed tightly, and left until the development process is complete.Lift the plate and then air dry.Observe the plate under 254 nm UV light, record, and bookmark.Calculate the Rf value of each spot (Wulandari et al., 2013).

Process Validation
Cut the GF 254 silica gel plate with a 5 x 10 cm size.Activate the plate at 1100C in the oven for 30 minutes.Make a line of 1.5 cm from the bottom, a creep distance of 8 cm, and an upper limit distance of 0.5 cm.Put the filter paper into the chamber, then add the motion phase of Chloroform -Methanol -Aquabidest with a ratio of 2:7:1 to the vessel and leave it until saturated.Put four marks on the line made at the bottom of the plate as a place to foul the solution B (Bc and Be) and C (Cc and Ce).
Point solution B (positive control) and solution C (comparator standard) on the plate using a capillary pipe.The plate is inserted into the saturated chamber, closed tightly, and left until the development process is complete.Insert the plate into the saturated chamber, then close it tightly and let it sit until the development process is complete.Lift the plate and then air dry.Observe the plate under 254 nm UV light, record, and mark.Calculate the Rf value of each spot (Wulandari et al., 2013).

Sample Identification by KLT
Cut GF 254 silica gel plates with 6 x 10 cm size.Turn the plate on at 110o C in the oven for 30 minutes.Make a line of 1.5 cm from the bottom, then a creep distance of 8 cm, and a distance of about 0.5 cm from the top border.Put the filter paper into the chamber, then add the motion phase of Chloroform -Methanol -Aquabidest with a ratio of 2:7:1 and leave it until saturated.
Mark the sample nodulations on the line on the plate's bottom at 5 points (A, Bc, Be, Cc, and Ce).Using a capillary pipe, point test solution A (sample), test solution B (positive control), and solution C (comparison standard) according to the point made on the plate.
Insert the plate into the saturated chamber, then close it tightly and let it sit until the development process is complete.Lift the plate and air dry it.Observe the plate under 254 nm UV light, record and mark it, and calculate the Rf value of each spot (Wulandari et al., 2013).

Sample Collection
Samples of shortness-of-breath herbal medicine were collected from each herbal medicine depot, which was either in the form of shophouses, tents, or mobile herbal medicine sellers in several areas of Cirebon City and Regency.

Organoleptic and Packaging Observations
As Table 2 shows, not all herbal samples meet the requirements related to the rules set by the Ministry of Health.The registration number is listed on the packaging in samples I, II, and III, as in Table II.However, after checking the BPOM website, the herbal medicine sample with the registration number was not registered.

Motion Phase Orientation
The research began by orienting the motion phase to find the composition of the motion phase that is good for the medicinal chemicals used, namely CTM and Ephedrine HCl.
At the orientation of the first phase of motion, the CTM spots are visible, but the Ephedrin HCL spots are very faint.However, if calculated, the Rf value of the two spots met the chromatogram efficiency parameters, which were 0.25 for CTM spots and 0.625 for Ephedrine HCl spots.
In the orientation of the second phase of motion, orientation is carried out to Ephedrine HCL only.Table 4 shows that Ephedrine HCL patches are still faint, even though a change of motion phase has been made.Even Ephedrine HCl patches are seen to spread.The spread of Ephedrine HCl spots is indicated because the carrier of the injection is water with very high polarity, which causes easy spread.
Then, the orientation of the third phase of motion was carried out with the latest composition, and thickening was carried out for the C Ephedrine HCl solution.In

Process Validation
Process validation is carried out to find an extraction procedure that attracts all the medicinal chemicals to be studied.Therefore, it can be concluded that the patch of solution B contains CTM and Ephedrine HCl.

Sample Identification
At the stage of sample identification of these two medicinal chemicals, the results are obtained as in the following table 5: In

Figure 1 .
Figure 1.Process Validation Chromatogram B: CTM and Ephedrine HCl Positive Control Solution (Simultaneous) Cc: CTM Comparator Solution Ce: Ephedrine HCl Comparator Solution identifying this shortness of breath herbal sample, observations were made on the chromatogram produced by comparing the color and hRf value of the test solution (A) on each sample with the positive control hRf (B).At the time of fouling, spots on solution B (positive control) should appear, then spots on solution A (samples) compared to spots on solution B (positive control).The sample is said to be positive if the hRf of solution A and B is the same or the difference must be less than 10%.Based on the description in Table5and some of the figures below, it can be concluded that 5 of the asthma herbal samples contain medicinal chemicals.

Table 1 . Distribution of Herbal Samples for Shortness of Breath
A: Breath drug (Asma) Head Cobra B: Blooming Tanjung Shortness of Breath C: Shortness of breath Cough Asthma Stamp Fruit Crown of God D : Asmagan Tribe 59 and Sesak Nafas Sabdo Palon F: Shortness of the breath of Tjipto's mother

Table 3 . Results of Organoleptic Observation of Herbal Samples
TableIVof the third phase of motion, the CTM and Ephedrine HCL spots are apparent.If calculated, the Rf value of the two spots meets the ideal chromatogram efficiency parameters, which are 0.31 for CTM and 0.71 for Ephedrine HCl.So, the phase of motion suitable for identifying CTM and Ephedrine HCl in this study is the composition of the mixed phase of motion from Chloroform -Methanolaquabidest with a ratio of 2:7:1.

Table 4 . Motion Phase Orientation Chromatogram
There is no difference between hRf spot B3e.2 and hRf C3e, and spot B3e.2 is Efedrin HCl.There is no difference between hRf spot A3.1 and hRf B3c.1, and spot A3.1 is CTM.The difference between hRf spot A3.2 and hRf B3e.2 is 1.43%, then the spot A3.2 is Ephedrine HCl.So it can be concluded that sample 3 is positive for CTM and Ephedrine HCl.