1. A method for separating and purifying flavonoids compounds in polygonum multiflorum leaves by high-speed counter-current chromatography is characterized by comprising the following steps:

(1) preparation of ethyl acetate phase of polygonum multiflorum

Drying polygonum multiflorum leaves, performing ultrasonic extraction by taking ethanol as a solvent, performing suction filtration, combining filtrate, performing vacuum concentration under reduced pressure to remove the solvent to obtain polygonum multiflorum leaf ethanol extract, adding ethyl acetate and water for multiple times of extraction, and performing vacuum concentration under reduced pressure to remove the solvent from an extracted ethyl acetate phase to obtain polygonum multiflorum leaf ethyl acetate phase;

(2) high-speed countercurrent chromatography separation and purification

Pumping a stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min by using a water-methanol-ethyl acetate-n-hexane solution with a volume ratio of 1:1:1: 0.5-2: 2:5:1 as a solvent system, pumping a mobile phase at a flow rate of 3-5 mL/min after the whole column is filled with the stationary phase, and simultaneously adjusting the rotation speed of the high-speed counter-current chromatograph to 800-900 rpm; when a two-phase solvent system reaches balance in a high-speed countercurrent column, dissolving the polygonum multiflorum ethyl acetate phase in the two-phase solvent system, wherein the sample injection concentration is 5-20 mg/mL, and the sample injection volume is 10-30 mL, and performing high-speed countercurrent chromatographic separation; and during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 200-300 nm, taking a single peak as a unit, collecting from the peak until the peak disappears, stopping collecting, respectively collecting different fractions, and performing reduced pressure concentration and freeze drying to obtain myricitrin, quercetin and kaempferol-3-O-rhamnoside.

2. The method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum thumb according to claim 1, wherein in the step (2), the volume ratio of water-methanol-ethyl acetate-n-hexane is 2: 1-3: 2-5: 0.5.

3. The method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum thumb according to claim 2, wherein in the step (2), the volume ratio of water-methanol-ethyl acetate-n-hexane is 2:1:3: 0.5.

4. The method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum thumb according to any one of claims 1 to 3, wherein in the step (2), when the high-speed countercurrent chromatography is performed, the rotation speed of a separation column is 850rpm, the flow rate of a mobile phase is 5mL/min, the wavelength of an ultraviolet detector is 254nm, the sample injection concentration is 11mg/mL, and the sample injection volume is 10 mL.

5. The method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum thumb according to claim 1, wherein in the step (1), the concentration of the ethanol solution extracted by ultrasonic is 80%.

6. The method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum by high-speed countercurrent chromatography according to claim 1, wherein in the step (1), the solid-to-liquid ratio of ultrasonic extraction is 1: 10-1: 20.

7. the method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum thumb according to claim 6 or 5, wherein in the step (1), the solid-to-liquid ratio of the ultrasonic extraction is 1: 15, the number of extractions was 2.

Background

Polygonum multiflorum (Polygonum multiflorum Thunb.), also known as caulis Polygoni Multiflori and caulis Polygoni Multiflori, is the dried tuber of Polygonum multiflorum (Polygonaceae) Polygonum multiflorum (Fallopia Adans.) of perennial herb. The records are recorded in compendium of materia Medica and Chinese pharmacopoeia, and the compendium of materia Medica says that the characters are sweet and flat; it enters heart and liver meridians. Has effects of nourishing blood, tranquilizing mind, dispelling pathogenic wind, and dredging collaterals. Can be used for treating insomnia, dreaminess, blood deficiency, general pain, rheumatic arthralgia, and skin pruritus. "the study shows that: the fleece-flower root contains various bioactive substances, wherein the most important substances comprise active ingredients such as flavonoid compounds, stilbene glycoside compounds, anthraquinone compounds, polyphenol compounds, phospholipids, fatty acids and the like. Modern pharmacological research shows that the fleece-flower root has the functions of tranquilizing and allaying excitement, resisting chronic inflammation, resisting bacteria and the like, and the flavonoid compound in the fleece-flower root also has the functions of resisting oxidation, resisting aging, resisting cancer and reducing high blood pressure, high blood fat and high blood sugar.

Researchers at home and abroad mostly concentrate on tuber roots and stems of polygonum multiflorum, and obtain that polygonum multiflorum tuber roots and stems contain a large amount of flavonoid compounds, stilbene glucoside compounds, polyphenol compounds, anthraquinone compounds, phospholipids, fatty acids and other chemical active ingredients [ polygonum multiflorum chemical ingredient and pharmacological action research progress, journal of Chinese experimental formulation chemistry, 2019, 13 th year ], but after tuber stems of polygonum multiflorum are removed, the rest of polygonum multiflorum is less researched (only polygonum multiflorum leaves and stems are researched, Chinese medicinal materials, 2009, 6 th year ], polygonum multiflorum leaves are researched for chemical ingredients and are separated to obtain flavonoids, steroids and glycosides and other compounds, the existing separation technology generally adopts traditional column chromatography separation, recrystallization and other methods to extract and separate monomeric compounds from polygonum multiflorum sample, and the methods need to be separated repeatedly, the operation is complex, the consumed time is long, the solvent consumption is large, the sample loss is large, and the separation effect is poor. High-speed counter-current chromatography is a new fast separation and chromatography separation technology in recent years, has the greatest advantage of no need of using a stationary phase as a carrier, has the unique advantages of small solvent dosage, high separation speed, large sample injection amount, high sample recovery rate and the like, and is widely applied to the preparation, separation and purification of phytochemistry, traditional Chinese medicine chemistry and natural product chemicals.

Disclosure of Invention

The invention aims to provide a method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum by high-speed counter-current chromatography, which can separate and purify a plurality of flavonoid compound monomers from the ethyl acetate phase of polygonum multiflorum.

In order to solve the technical problem, the invention is realized as follows:

the method for separating and purifying flavonoids compounds in the ethyl acetate phase of polygonum multiflorum comprises the following steps:

(1) preparation of ethyl acetate phase of polygonum multiflorum

Drying polygonum multiflorum leaves, and performing ultrasonic extraction for 1-3 times by taking ethanol as a solvent, wherein the extraction time is 1-4 h, and the solid-to-liquid ratio of the ultrasonic extraction is 1: 10-1: 20, carrying out suction filtration, combining filtrates, carrying out vacuum reduced pressure concentration to remove the solvent to obtain a polygonum multiflorum ethanol extract, then adding ethyl acetate and water to carry out extraction for multiple times, and carrying out vacuum reduced pressure concentration to the completely extracted ethyl acetate phase to remove the solvent to obtain a polygonum multiflorum ethyl acetate phase;

(2) high-speed countercurrent chromatography separation and purification

Pumping a stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min by using a water-methanol-ethyl acetate-n-hexane solution with a volume ratio of 1:1:1: 0.5-2: 2:5:1 as a solvent system, pumping a mobile phase at a flow rate of 3-5 mL/min after the whole column is filled with the stationary phase, and simultaneously adjusting the rotation speed of the high-speed counter-current chromatograph to 800-900 rpm; when a two-phase solvent system reaches balance in a high-speed countercurrent column, dissolving the polygonum multiflorum ethyl acetate phase in the two-phase solvent system, wherein the sample injection concentration is 5-20 mg/mL, and the sample injection volume is 10-30 mL, and performing high-speed countercurrent chromatographic separation; and during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 200-300 nm, taking a single peak as a unit, collecting from the peak until the peak disappears, stopping collecting, respectively collecting different fractions, and performing reduced pressure concentration and freeze drying to obtain myricitrin, quercetin and kaempferol-3-O-rhamnoside.

Particularly, the volume ratio of the water to the methanol to the ethyl acetate to the n-hexane is 2: 1-3: 2-5: 0.5.

In particular, in the step (2), the volume ratio of the water to the methanol to the ethyl acetate to the n-hexane is 2:1:3: 0.5.

Specifically, in the step (2), when the high-speed countercurrent chromatography separation is performed, the rotation speed of the separation column is 850rpm, the flow rate of the mobile phase is 5mL/min, the wavelength of the ultraviolet detector is 254nm, the sample injection concentration is 11mg/mL, and the sample injection volume is 10 mL.

Specifically, in the step (1), the ethanol solution obtained by ultrasonic extraction has a concentration of 80%.

In particular, in the step (1), the solid-liquid ratio of ultrasonic extraction is 1: 10-1: 20.

in particular, in the step (1), the solid-liquid ratio of the ultrasonic extraction is 1: 15, the number of extractions was 2.

The method can separate and purify three flavonoid monomers, namely myricitrin, quercitrin and kaempferol-3-O-rhamnoside, and can separate and purify the three flavonoid monomers from the ethyl acetate phase of polygonum multiflorum at one time, so that the separation and purification of the flavonoid monomers are high in efficiency, simple to operate, low in comprehensive cost, large in separation amount, high in product purity and small in sample loss; the purity of the flavonoid monomer obtained by separation and purification reaches more than 90 percent through High Performance Liquid Chromatography (HPLC), and the purity of the myricitrin, the quercitrin and the kaempferol-3-O-rhamnoside of the flavonoid monomer can be more than 95 percent through the purification of each fraction through preparative Liquid Chromatography.

Drawings

FIG. 1 is a chromatogram of a high-speed countercurrent chromatography separation and purification of ethyl acetate phase of polygonum multiflorum by using water-methanol-ethyl acetate-n-hexane in a volume ratio of 2:1:3:0.5 as a solvent system;

FIG. 2 is a high performance liquid chromatogram of separated and purified myricitrin;

FIG. 3 is a high performance liquid chromatogram of separated and purified quercetin;

FIG. 4 is a high performance liquid chromatogram of kaempferol-3-O-rhamnoside separated and purified.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The method for separating and purifying the flavonoid compounds in the ethyl acetate phase of the polygonum multiflorum provided by the invention is described in detail as follows:

the method comprises the following steps: preparation of ethyl acetate phase of polygonum multiflorum

Carrying out ultrasonic extraction on 1.0kg of dried polygonum multiflorum leaves by taking 90% ethanol as a solvent, wherein the extraction time is 2h, and the solid-to-liquid ratio of the ultrasonic extraction is 1: 15, filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain ethanol extract, and extracting with ethyl acetate and water for several times. And (3) carrying out vacuum reduced pressure concentration on the ethyl acetate phase after complete extraction to remove the solvent, thus obtaining the polygonum multiflorum ethyl acetate phase.

Step two: flavonoid monomer separation

The method comprises the steps of taking water-methanol-ethyl acetate-n-hexane with a volume ratio of 1:1:1: 0.5-2: 2:5:1 as a solvent system, fully shaking up, standing, separating upper and lower phases, taking the upper phase as a stationary phase and the lower phase as a mobile phase, performing ultrasonic degassing, injecting the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min, injecting the mobile phase at a flow rate of 3-5 mL/min after the whole column is filled with the stationary phase to form a two-phase solvent system, and adjusting the rotation speed of the counter-current chromatograph to 700-900 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the polygonum multiflorum ethyl acetate in the two-phase solvent system to prepare a separation sample, introducing the separation sample into a High-Speed countercurrent chromatograph, wherein the concentration of the separation sample is sample introduction concentration, the sample introduction concentration is 5-20 mg/mL, the sample introduction volume is 10-30 mL, and separating the polygonum multiflorum ethyl acetate phase by High-Speed Counter-Current Chromatography (HSCCC); during high-speed counter-current chromatographic separation, an ultraviolet detector monitors on line, the detection wavelength is 200-300 nm, corresponding peak components are respectively collected according to chromatographic peaks, and the corresponding high-purity compound is obtained by decompression, concentration and drying;

step three: purity determination and structure determination of flavonoid monomer

The collected fractions are respectively passed through¹H-NMR (Nuclear Magnetic Resonance) and¹³C-NMR identification.

Example 1

The method comprises the following steps: preparation of ethyl acetate phase of polygonum multiflorum

Carrying out ultrasonic extraction on 1.0kg of dried polygonum multiflorum leaves by taking 90% ethanol as a solvent, wherein the extraction time is 2h, and the solid-to-liquid ratio of the ultrasonic extraction is 1: 15, filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain ethanol extract, and extracting with ethyl acetate and water for several times. And (3) carrying out vacuum reduced pressure concentration on the ethyl acetate phase after complete extraction to remove the solvent, thus obtaining the polygonum multiflorum ethyl acetate phase.

Step two: flavonoid monomer separation

Adopting water-methanol-ethyl acetate-n-hexane with a volume ratio of 2:1:3:0.5 as a solvent system, fully shaking up, standing, separating an upper phase from a lower phase, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonically degassing, injecting the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20mL/min, injecting the mobile phase at a flow rate of 5mL/min after the whole column is filled with the stationary phase to form a two-phase solvent system, and adjusting the rotating speed of the counter-current chromatograph to 850 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the polygonum multiflorum ethyl acetate in the two-phase solvent system to prepare a separation sample, introducing the separation sample into a High-Speed countercurrent chromatograph, wherein the concentration of the separation sample is sample introduction concentration, the sample introduction concentration is 11mg/mL, the sample introduction volume is 10mL, and the polygonum multiflorum ethyl acetate phase is subjected to High-Speed Counter-Current Chromatography (HSCCC) separation; during high-speed counter-current chromatography, an ultraviolet detector is used for on-line monitoring, the detection wavelength is 254nm, corresponding peak components are respectively collected according to chromatographic peaks, and the corresponding high-purity compound is obtained by reduced pressure concentration and drying, as shown in figure 1.

Myricitrin (I), quercitrin (II) and kaempferol-3-O-rhamnoside (III).

Step three: purity determination and structure determination of flavonoid monomer

The collected fractions are respectively passed through¹H-NMR (Nuclear Magnetic Resonance) and¹³C-NMR identification, wherein the obtained monomeric compounds are 30.93mg of myricitrin, 23.2mg of quercitrin and 13.38mg of kaempferol-3-O-rhamnoside respectively; and (3) respectively detecting the collected fractions by HPLC (high performance liquid chromatography), calculating by using a chromatographic peak area normalization method, measuring the area of each impurity peak and the total chromatographic peak area, calculating the percentage of each impurity peak area and the sum of the impurity peak areas in the total peak area, and obtaining the sample purity, namely the percentage of the removed impurity peak sum. As shown in fig. 2, the peak area of the impurity peak is 5.1% and the total chromatographic peak area is 1 as calculated by the chromatographic peak area normalization method, the purity of the obtained myricitrin is 94.9%, the purity of the quercetin is 97.0% and the purity of the kaempferol-3-O-rhamnoside is 95.7% as known from the same principle; the high performance liquid chromatogram of myricitrin, quercetin and kaempferol-3-O-rhamnoside are shown in fig. 2-4 respectively.

Example 2

The method comprises the following steps: preparation of ethyl acetate phase of polygonum multiflorum

Carrying out ultrasonic extraction on 1.0kg of dried polygonum multiflorum leaves by taking 85% ethanol as a solvent, wherein the extraction time is 1h, and the solid-to-liquid ratio of the ultrasonic extraction is 1: 20, filtering, and repeatedly treating filter residues for 1 time; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain ethanol extract, and extracting with ethyl acetate and water for several times. And (3) carrying out vacuum reduced pressure concentration on the ethyl acetate phase after complete extraction to remove the solvent, thus obtaining the polygonum multiflorum ethyl acetate phase.

Step two: flavonoid monomer separation

Adopting water-methanol-ethyl acetate-n-hexane with a volume ratio of 2:2:4:0.5 as a solvent system, fully shaking up, standing, separating an upper phase from a lower phase, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonically degassing, injecting the stationary phase into a high-speed counter-current chromatograph at a flow rate of 25mL/min, injecting the mobile phase at a flow rate of 4mL/min after the whole column is filled with the stationary phase to form a two-phase solvent system, and adjusting the rotating speed of the counter-current chromatograph to 800 rpm; when the two-phase solvent system reaches dynamic balance in the countercurrent column, dissolving the polygonum multiflorum ethyl acetate in the two-phase solvent system to prepare a separation sample, feeding the separation sample into a high-speed countercurrent chromatograph, wherein the concentration of the separation sample is sample introduction concentration, the sample introduction concentration is 15mg/mL, the sample introduction volume is 20mL, and performing HSCCC separation on the polygonum multiflorum ethyl acetate phase; during high-speed counter-current chromatographic separation, an ultraviolet detector is used for on-line monitoring, the detection wavelength is 270nm, corresponding peak components are respectively collected according to chromatographic peaks, and the corresponding high-purity compound is obtained by reduced pressure concentration and drying.

Step three: purity determination and structure determination of flavonoid monomer

The purity of the monomeric compound is detected by high performance liquid chromatography as in step three of the embodiment 1.

Example 3

The method comprises the following steps: preparation of ethyl acetate phase of polygonum multiflorum

Carrying out ultrasonic extraction on 1.0kg of dried polygonum multiflorum leaves by using 95% ethanol as a solvent for 3h, wherein the solid-liquid ratio of the ultrasonic extraction is 1: 10, filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain ethanol extract, and extracting with ethyl acetate and water for several times. And (3) carrying out vacuum reduced pressure concentration on the ethyl acetate phase after complete extraction to remove the solvent, thus obtaining the polygonum multiflorum ethyl acetate phase.

Step two: flavonoid monomer separation

Adopting water-methanol-ethyl acetate-n-hexane with a volume ratio of 2:1:4:1 as a solvent system, fully shaking up, standing, separating an upper phase from a lower phase, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonically degassing, injecting the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20mL/min, injecting the mobile phase at a flow rate of 3mL/min after the whole column is filled with the stationary phase to form a two-phase solvent system, and adjusting the rotating speed of the counter-current chromatograph to 900 rpm; when the two-phase solvent system reaches dynamic balance in the countercurrent column, dissolving the polygonum multiflorum ethyl acetate in the two-phase solvent system to prepare a separation sample, feeding the separation sample into a high-speed countercurrent chromatograph, wherein the concentration of the separation sample is the sample injection concentration, the sample injection concentration is 20mg/mL, the sample injection volume is 20mL, and performing HSCCC separation on the polygonum multiflorum ethyl acetate phase; during high-speed counter-current chromatographic separation, an ultraviolet detector is used for on-line monitoring, the detection wavelength is 230nm, corresponding peak components are respectively collected according to chromatographic peaks, and the corresponding high-purity compound is obtained by reduced pressure concentration and drying.

Step three: purity determination and structure determination of flavonoid monomer

The purity of the monomeric compound is detected by high performance liquid chromatography as in step three of the embodiment 1.

Example 4

The method comprises the following steps: preparation of ethyl acetate phase of polygonum multiflorum

Carrying out ultrasonic extraction on 1.0kg of dried polygonum multiflorum leaves by taking 80% ethanol as a solvent, wherein the extraction time is 3h, and the solid-to-liquid ratio of the ultrasonic extraction is 1: 20, filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain ethanol extract, and extracting with ethyl acetate and water for several times. And (3) carrying out vacuum reduced pressure concentration on the ethyl acetate phase after complete extraction to remove the solvent, thus obtaining the polygonum multiflorum ethyl acetate phase.

Step two: flavonoid monomer separation

Adopting water-methanol-ethyl acetate-n-hexane with a volume ratio of 2:1:5:0.5 as a solvent system, fully shaking up, standing, separating an upper phase from a lower phase, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonically degassing, injecting the stationary phase into a high-speed counter-current chromatograph at a flow rate of 15mL/min, injecting the mobile phase at a flow rate of 3mL/min after the whole column is filled with the stationary phase to form a two-phase solvent system, and adjusting the rotating speed of the counter-current chromatograph to 800 rpm; when the two-phase solvent system reaches dynamic balance in the countercurrent column, dissolving the polygonum multiflorum ethyl acetate in the two-phase solvent system to prepare a separation sample, feeding the separation sample into a high-speed countercurrent chromatograph, wherein the concentration of the separation sample is the sample injection concentration, the sample injection concentration is 20mg/mL, the sample injection volume is 10mL, and performing HSCCC separation on the polygonum multiflorum ethyl acetate phase; during high-speed counter-current chromatographic separation, an ultraviolet detector is used for on-line monitoring, the detection wavelength is 300nm, corresponding peak components are respectively collected according to chromatographic peaks, and the corresponding high-purity compound is obtained by reduced pressure concentration and drying.

Step three: purity determination and structure determination of flavonoid monomer

The purity of the monomeric compound is detected by high performance liquid chromatography as in step three of the embodiment 1.

Example 5

The method comprises the following steps: preparation of ethyl acetate phase of polygonum multiflorum

Carrying out ultrasonic extraction on 1.0kg of dried polygonum multiflorum leaves by using 75% ethanol as a solvent for 2.5h, wherein the solid-liquid ratio of the ultrasonic extraction is 1: 10, filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain ethanol extract, and extracting with ethyl acetate and water for several times. And (3) carrying out vacuum reduced pressure concentration on the ethyl acetate phase after complete extraction to remove the solvent, thus obtaining the polygonum multiflorum ethyl acetate phase.

Step two: flavonoid monomer separation

Adopting water-methanol-ethyl acetate-n-hexane with a volume ratio of 2:1:2:0.5 as a solvent system, fully shaking up, standing, separating an upper phase from a lower phase, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonically degassing, injecting the stationary phase into a high-speed counter-current chromatograph at a flow rate of 30mL/min, injecting the mobile phase at a flow rate of 4mL/min after the whole column is filled with the stationary phase to form a two-phase solvent system, and adjusting the rotating speed of the counter-current chromatograph to 820 rpm; when the two-phase solvent system reaches dynamic balance in the countercurrent column, dissolving the polygonum multiflorum ethyl acetate in the two-phase solvent system to prepare a separation sample, feeding the separation sample into a high-speed countercurrent chromatograph, wherein the concentration of the separation sample is sample introduction concentration, the sample introduction concentration is 15mg/mL, the sample introduction volume is 25mL, and performing HSCCC separation on the polygonum multiflorum ethyl acetate phase; during high-speed counter-current chromatographic separation, an ultraviolet detector is used for on-line monitoring, the detection wavelength is 200nm, corresponding peak components are respectively collected according to chromatographic peaks, and the corresponding high-purity compound is obtained by reduced pressure concentration and drying.

Step three: purity determination and structure determination of flavonoid monomer

The purity of the monomeric compound is detected by high performance liquid chromatography as in step three of the embodiment 1.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.